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Historic, archived document 

Do not assume content reflects current 
scientific knowledge, policies, or practices 



ARS 74—47 
November 1968 



U.S. DEFT. OF AGJ^i'CULTURE 

JUL 1 ]9bhJ 

CURRENT SERiAUECOfiOS 



MOLECULAR BIOLOGY 
AND AGRICULTURE 

Potential for Future Research 



Report of 
Western Experiment Station 
Collaborators Conference 
March 13—16, 1968, 

Albany, California 




Agricultural Research Service 
United States Department of Agriculture 



1 



EACH YEAR THE REGIONAL UTILIZATION RESEARCH DIVISIONS OF THE 
U.S. Department of Agriculture hold conferences attended by 
representatives of State Agricultural Experiment Stations in 
their regions. 

These conferences of agricultural scientists fulfill 
needs for reports and discussion in areas of special impor- 
tance. In the western region the subject in 1967 was 
Proteins for Aid and Trade; in 1966 it was The Destruction 
of Salmonellae. 

In 1968 (March 13-15) the subject was Molecular Biology 
in Agriculture: Potential for Future Research. Molecular 
biology is in certain respects a new scientific discipline, 
created within the past two decades by deeply penetrating 
biochemical discoveries in the area of genetic processes. 
Within the U.S. Department of Agriculture this conference 
was the first to be exclusively devoted to molecular biology. 

The program was planned by Alfred C. Olson, Chemist in 
the Plant Enzyme Pioneering Laboratory of the Western Utiliz- 
ation Research and Development Division, and Paul K. Stumpf, 
Chairman of Biochemistry and Biophysics in the University of 
California at Davis. 

The plan used in reporting the conference is explained 
in a note on the following page. Occasional use of commercial 
names for materials does not imply reconmendation by the U.S. 
Department of Agriculture. 

The data, illustrations, references, and the style used 
in references are those supplied by the speakers. 

This report was produced in the Division listed below, 
and copies are available from that source. 



Western Utilization Research and Development Division 
Agricultural Research Service 
UNITED STATES DEPARTMENT OF AGRICULTURE 
Albany, California 94710 



CONTENTS 

Some explanation of the arrangement of materials in this report 
is necessary. Those who planned the • conference realized that 
the contributors are scientists engaged in research in segments 
of the general area of molecular biology. Their contributions, 
which are presented in summarized form, reveal problems and 
discuss research methods and advancements. 

The need for discussion directed toward review and perspective 
also seemed apparent. This need has been appropriately met by 
remarks authored by James Bonner, Professor of Biology in the 
California Institute of Technology, and presented by him 
informally. Salient extracts from his remarks are included 
here. For convenience, they are placed first. 

, Page 



Background and Outlook, James Bonner ------------- 3 

The Conformation of Nucleic Acids in Solution, . 

John T. Marvel 8 

Templates in a Test Tube: Enzymatic Replication 

of DNA, Slgmund Schwimmer -----------------11 

ENA Transcription, J. S. Krakow ---------------14 

Regulatory Mechanisms Involving Enzyme Function, 

Synthesis and Degradation, Philip Filner ----------17 

Cellular Organization, E. H. Mercer -------------21 

Chloroplast Structure and Development, 

W. M. Laetsch 26 

Some Aspects of the Molecular Biology of Chloroplasts , 

S. G. Wildman ■ 31 

Bacterial Sporulation: A Model System for the Molecular 

Approach to Morphogenesis, Roy H. Doi -----------35 

Nucleic Acids in Evolution, Taxonomy and Development, 

B. J. McCarthy 38 

Leaf. Abscission: The Chronology and Control of a 

Terminal Developmental Sequence, D. James Morre ------ 39 

Metabolic and Physiological Development in Plant Tissues, 

George G. Laties ----------------------43 

Attendance --------------------------45 



MOLECULAR BIOLOGY AND AGRICULTURE 

POTENTIAL FOR FUTURE RESEARCH 



BACKGROUND AND OUTLOOK 
James Bonner 

Department of Biology, California Institute of Technology 

(Two portions of Dr. Bonner's remarks are included below. 
The first one is a statement of present status. The second 
is a longer excerpt from remarks that were recorded on tape 
and transcribed.) 

Present Status 

During the first half of this century, two of the major 
branches of agricultural research, breeding and utilization, have 
been nourished and guided by the sciences of genetics and bio- 
chemistry, respectively. Just as the new science of molecular 
biology has wedded these two disciplines into a penetrating 
understanding of life, so may we expect that it will erase all 
barriers between them. 

At the core of molecular biology is DNA (deoxyribonucleic 
acid), the genetic substance. Its role as the master template 
for the functioning and reproduction of cells of living organisms 
is now well established. Molecular biology tells us that the 
synthesis of enzymes required for this functioning is under the 
control of, and is dictated by, the sequence of nucleotides in 
the DNA. 

Less well known is the method by which cells differentiate 
and become specialized. Although all cells of a given organism 
contain the same genetic code (the same DNA) the expression of 
much of the code in the cell of a given organ at a given time is 
guided by represser substances. In bacteria these repressers are 
specific proteins which combine with those specific regions of 
DNA that are to be repressed. In higher organisms such as plants, 
the repression is effected by nonspecific proteins, histones, to 
which are attached short RNA (ribonucleic acid) chains. Recent 
research indicates that specificity of repression lies in the 
nucleotide sequence of this histone-associated RNA. Histones can 
be selectively removed from the DNA, thus "turning on" specific 
genes . 



- 3 - 



Thus the pathway of differentiation and development of a 
given cell is dependent upon its local environment. Recent 
experiments show that development and differentiation can be 
artificially influenced (i.e., the chromosomes can be reprogram- 
ed) . These investigations suggest new ways to endow plants with 
desired genetic characteristics, the possibility, of creating a 
completely new plant species, or a way to make identical copies 
of one plant. Such novel and still unachieved approaches do not 
mean that advancement in plant and animal breeding by traditional 
means will soon be exhausted. Dwarf wheat and high-lysine corn 
are results of traditional methods and undoubtedly more similarly 
excellent achievements will be forthcoming. 

Background and Potential 

A cell lives and multiplies because within that cell is a 
book of instructions on how to make everything that's in that cell 
This "book" is DNA or deoxyribonucleic acid. It is an extremely 
large molecule made of four kinds of building blocks or nucleo- 
tides called adenine, thymine, guanine, and cytosine, commonly 
designated by their initials. A, T, G, and C. DNA is able to 
replicate and it's the only molecule that has that power. 

One of the greatest triumphs of modern biology was the 
fairly recent discovery of the mode of replication of DNA. Each 
molecule consists of two chains wrapped around one another. The 
first is mated with a companion chain, also made of A, T, G, and C 
The two chains are mated in accord with a special law called 
complementarity. This law says that where there's an A in chain 
No. 1, there must be a T in chain No. 2. Where there's a T in 
chain No. 1, there must be an A in chain No. 2. Where there's a 
G in chain No. 1, there must be a C in chain No. 2. And where 
there's a C in chain No. 1, there has to be a G in chain No. 2. 
Each cell contains a pool of the nucleotides. The two chains 
draw on the pool. When the monomers have piled up on the tem- 
plates the DNA polymerase connects them. The chains separate 
and we have two molecules, each identical with the original. Now 
the cell is free to divide and give to each daughter a copy of 
the genetic information. 

All other parts of the cell are concerned with providing 
a supply of A, T, G, and C. The DNA causes A, T, G, and C to be 
made by enzymes which use the available food. DNA also makes 
another kind of long-chain molecule, called ribonucleic acid or 
RNA. RNA consists of four kinds of building blocks that are 
analogous to A, T, G, and C. They are called A', U' , G', and C'. 
The U stands for uracil, which resembles thjrmine. The cell 
contains a pool of these building blocks. In their presence. 



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the double-stranded DNA becomes temporarily single stranded and 
the monomers align themselves along the single-stranded chain. U' 
pairs with A, A' pairs with T, C' pairs with G, G' pairs with C. 

When that assemblage is completed and the monomers are 
arranged on the DNA template, an enzjnne, a second kind of poljnner- 
ase, connects them. We now have an RNA molecule. Then the 
hydrogen bonds which held the RNA to the DNA break. Those that 
held DNA together are reestablished and the RNA molecule is now 
free. RNA molecules cannot replicate themselves. They have one 
great power which DNA molecules do not have. RNA molecules can 
direct the synthesis of enzjmie molecules. 

How does this work? The DNA molecule is long, correspond- 
ing to a million "letters" or more. The RNA molecules are short. 
The RNA represents the information in one genetic unit of the DNA. 
It is a small section of the DNA, corresponding to one or two 
thousand letters. The "letters" are coded instructions on how to 
make a single kind of enzyme molecule. One kind of RNA transcribed 
from DNA, for example, will direct the synthesis of peroxidase 
while another will direct the synthesis of a-amylase. 

How is this information used? An enzyme molecule is made of 
amino acids strung together in a linear chain. The kind of enzyme 
is determined by the sequence of the 20 kinds of amino acid mono- 
mers. The enzyme molecule, on the average, is a hundred or a few 
hundred amino acids long, and contains the message units necessary 
to synthesize enzymes, including directions to start the synthesis, 
to add amino acids in a definite pattern, and finally to terminate 
the molecule. 

The RNA message is translated by a large enzyme molecule, a 
super enzjmie molecule, called a ribosome. There are about 5-50 
million ribosomes in a typical cell. The messenger RNA molecule 
fastens to the ribosome, which then translates each message unit. 
It moves bodily down the messenger RNA molecule reading what it 
says, putting things together, and when it comes to the end it 
sends the finished enzyme out to work. In a typical cell of a 
higher organism like ourselves, there are about ten thousand 
kinds of enzyme molecules and there are, on the average, about 
50 thousand individuals of each kind. Thus only one kind is a 
very small fraction of the total. 

During the past several years, it has become possible to 
isolate the chromosomes from higher organisms. When isolated 
chromosomes are supplied with A', U', G', and C' and the poljmier- 
ase enz3mie, they make messenger RNA molecules just as they do in 
life. And it is possible to show by experiment that in isolated 
chromosomes the same genes are turned on for the making of 



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messenger RNA, as in life, and the same genes are also turned off 
as in life. Using isolated chromosomes we can try to find what it 
is that turns on some genes and turns other off. 

We know what the material basis is. We know that of the 
many kinds of proteins in a cell, there is one whose duty is to be 
wrapped around the DNA. This kind of protein is found only in the 
chromosomes of higher organisms. Everything that has a nucleus 
has these proteins, which are known as histones. When DNA has 
histone protein wrapped around it, the poljmierase enzjmie cannot 
transcribe it, cannot make a messenger RNA molecule. Thus in the 
cells of one part of a differentiated creature, certain parts of 
the DNA are not covered with histone. Here RNA and enzymes can 
be made. In another part, these portions of the DNA are covered 
with histone and cannot be transcribed and the genes are turned off. 

Very recently we found out how to remove histones from the 
DNA. The molecules of histone are attached to the DNA by a little 
piece of nucleic acid. Proteins can't read the sequence of 
letters in a DNA very well. They must read the letters by a kind 
of base pairing, or base complementarity. Each of the protein 
molecules that interacts with a specific gene of DNA to repress 
it has a little piece of RNA which, by the strategy of U' pairing 
with A, A' pairing with T, C' pairing with G, etc., finds its 
complementary place in the DNA, and sits down and deposits histone 
molecules on the gene. These RNA molecules are 40 nucleotides 
long in plants and peas and about 60 nucleotides long in rats, 
cows, and humans. We can separate the histone molecules from the 
associated DNA of one organ and make a solution of them. We can 
also take purified DNA from a different organ of the same organism 
and mix the two — put them back together. For example, we can take 
liver histone proteins and muscle DNA, mix them, let them rejoin, 
and thus make chromosomes that are identical with the liver 
chromosomes. Now we are learning something about the way in which 
DNA molecules are specifically repressed. We have a long way to 
go to find out how nature removes histones and puts them back but 
we're making progress. 

Let's consider next how differentiation might occur. When 
we take some cells from a higher organism, like a carrot, and grow 
them in tissue culture, they grow just like bacterial cells. If 
we take a single cell from such a culture and put it in nutrient 
medium containing chemical substances that are normally in the 
embryo sac, or the sac in which plant embryos normally develop, 
the cell develops into an embryo, and ultimately into a whole 
plant. Thus single cells multiply and develop into embryos when 
they are placed in the appropriate media. In this way one can 
make literally millions of embryos or millions of adult plants 



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with exactly the original constitution. We now have a way of 
resetting the developmental program of a mature plant cell that 
would normally never divide again. 

We can't do this with animal cells yet. A related thing, 
however, can be done with animals. The nucleus of the egg of a 
Xenopus toad can be either removed or inactivated. The nucleus 
can be scooped out of the egg or killed with an ultraviolet beam 
so that it can't work. Next we take a specialized cell from a 
Xenopus tadpole, such as a cell from the epithelial lining of 
the gut. This adult cell, containing a nucleus, would normally 
never divide again although it still makes RNA, ribosomes, and 
enzymes. The nucleus of this cell can be transplanted into the 
previously prepared egg. Within a few minutes it starts to 
behave like the nuclei in the tissue culture experiment I 
mentioned before. The nucleus "discovers" that the egg contains 
compounds that make this an egg. The compounds and cytoplasm of 
the egg interact with the represser molecules and the DNA of the 
chromosomes of the transplanted nucleus and bring about the 
situation of genes turned on and genes turned off, appropriate 
for a fertilized egg. This egg now develops into an adult toad. 
Thus, we can make any number of creatures that are identical in 
genetic constitution. 

I don't doubt that this kind of information will be useful 
in making new plants and animals. By breeding procedures it is 
impossible to make animals of identical genetic constitution. It 
is now possible to use a kind of vegetative reproduction with 
animals as we have with some kinds of plants, for example, date 
palms, for thousands of years. These simple examples show that 
we can reprogram the chromosomes of an adult organism to fertil- 
ized egg time. With increasing knowledge we may be able to 
reprogram the chromosomes of an adult cell to any desired spot 
in the developmental history — or into new lungs or hearts or 
other desired organs. 

You might inquire whether molecular biology has been able 
to transplant a chromosome or a piece of a chromosome from one 
organism into a second acceptor, as a means of transferring genetic 
information. This has been possible so far only with the bacteria. 

These brief remarks provide us with a background. As our 
understanding of these basic processes increases, so will gains be 
made in applied agriculture. The dramatic successes of plant and 
animal breeding programs of recent years might be surpassed if 
experiments currently in progress in making crosses between 
species prove successful. In this way I would envisage for example 



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the addition of the nitrogen fixation character to, let us say, 
the cereal grains. In this new technology we have for the first 
time a way to get around nature's barrier to crosses. 

RELATED READING - 

Wooldridge, Dean. 1966. The Machinery of Life. McGraw-Hill Book 
Company, New York. 

Beadle, George and Muriel. 1966. The Language of Life. Doubleday 
and Company, Garden City, New York. 

Bonner, James and J. E. Varner, eds.. Plant Biochemistry . 1965. 
Academic Press, New York. 

Bonner, James. 1965. The Molecular Biology of Development. 
Oxford University Press, New York. 

Watson, J. D. 1965. Molecular Biology of the Gene. 
W. A. Benjamin, Inc., New York, Amsterdam. 



THE CONFORMATION OF NUCLEIC ACIDS IN SOLUTION 

John T. Marvel 
Department of Agricultural Biochemistry 
University of Arizona, Tucson 

The important primary, secondary, and tertiary features of 
DNA have been known for a considerable time (1) and an extensive 
knowledge of the same features for RNA has developed in the past 
decade (2). Studies of conformation or of conformational changes 
of nucleic acids have been made using almost every known physical 
and biological technique (3). The four factors which contribute 
to the conformations of nucleic acids are: the structure of the 
monomeric unit, hydrogen bonding, base stacking, and the chemical 
environment (i.e., the solvents and ions present). 

A recent article by Haschemeyer and Rich (4) considers con- 
formation of nucleosides theoretically by calculation of the 
contact interactions of the atoms through use of crystal log raphic 
data for their bond lengths. Under consideration is whether the 



- 8 - 



heterocyclic base is syn or anti with respect to a plane passing 
perpendicularly through the C-, ~ 0^, bond; whether the or C^t 
atom is out of plane and in what sense, exo or endo. These 
workers concluded that the predominant form for the major nucleo- 
tides is anti, in agreement with other work (5). No conclusion 
was reached concerning the furanose ring conformation, even though 
only one conformation should be allowed in a biologically active 
macromolecule . 

Model systems of di- and tri-nucleotldes have been used to 
demonstrate hydrogen bonding (6) and base stacking. Nuclear 
magnetic resonance has been used recently in these fields (8), 
and provides one of the most graphic examples of stacking in the 
"near neighbor effects" shown by the thymidine methyl group (8). 
Studies of the environmental factors influencing oligo nucleotide 
ordering are now receiving much attention (9,10). Fresco et al. 
have been able to correlate conformation changes in s-RNA with 
amino acid acceptor activity, and to show the great importance 
of metal ions to the active conformation (2b). The melting 
curves obtained by these workers for s-RNA show an initial melt 
between 20* and 40° when monitored by sedimentation value or 
viscosity, which does not appear when monitored by CD. changes. 
It is interesting that the n.m.r. data of McDonald, Phillips, and 
Penswick (8c) would be in accord with the former results. High- 
molecular-weight RNA (and single-stranded DNA) has been postulated 
to have organized double stranded helical regions and disorganized 
loops or folds (2a). That organized structure is present even in 
synthetic polynucleic acids is clearly seen in the work of 
Michelson' and associates (10) and other workers using synthetic 
and chemically modified polynucleotides (11). Michelson can 
demonstrate differential reactivity of polynucleotides, depending 
on whether they are single or double stranded, and by changing 
the reaction conditions. The effect of chemicals on the confor- 
mation of nucleic acids in solution has special importance in 
mutagenic studies involving TMV, as has been recognized by Singer 
and Fraenkel-Conrat (12a). This is easily seen by the variety of 
conditions TMV-RNA is subjected to in mutagenic studies (12a, b,c). 
A clear case of conformational effects involving TMV-RNA can be 
shown by chain-breakage studies involving the reaction of non- 
aqueous diazomethane (13). TMV-RNA stays largely intact until a 
very large excess of diazomethane is employed; then it is 
extensively degraded. In view of the facile degradation of 
dinucleotide phosphates by diazomethane (14) it is surprising 
that TMV-RNA remains intact as long as it does. This appears 
to be a case where conformation is stabilizing the nucleic acid. 



- 9 - 



Literature Cited 

1. J. D. Watson and F. H. C. Crick. Nature 171: 737, 964 (1963). 

2. (a) A. S. Spirin, Progress in Nucleic Acid Research, J. N. 
Davidson and W. E. Cohen (eds.). Academic Press, New York, 
Vol. 1, p. 301 (1963). (b) J. R. Fresco, A. Adams, R. Ascine, 
D. Henley, and T. Lindahl. Cold Springs Harbor Symp. Quant. 
Biol. 31: 527 (1966). (c) M. Leng and A. M. Michelson. 
Biochim. et Biophys. Acta. 155 : 91 (1968) and references 
contained therein. 

3. H. R. Maples and E. H. Cordes. Biological Chemistry, Harper 
& Row, New York, pp. 143-180 (1966). 

4. A. E. V. Haschemeyer and A. Rich. J. Mol. Biol. 27_: 369 (1967). 

5. (a) M. Sundaralingam. J. Am. Chem. Soc. 87: 599 (1965). 

(b) D. W. Miles, R. K. Rabins et al. J. Phys. Chem. 71_: 3931 
(1967). 

6. (a) R. R. Shoup, H. T. Miles, and E. D. Becker. Biochem. 
Biophys. Res. Commun. _23: 194 (1966). (b) S. Lunell and G. 
Sperber. J. Chem. Phys. 26: 2119 (1967). (c) A. Novak and 
A. Lamtie. Nature 216: 1202 (1967). (d) R. C. Lord and 

G. J. Thomas, Jr. Biochim. et Biophys. Acta 142 ; 1 (1967). 

(e) Y. Kyogoku, R. C. Lord et al. Science 154: 518 (1966). 

(f) J. Brahns and Ch. Sadron. Nature 212: 1389 (1966). 

7. (a) C. R. Cantor and I. Tinoco, Jr. Biopolymers 5.: 821 (1967). 

(b) G. J. Thomas, Jr. and Y. Kyogoku. J. Am. Chem. Soc. 89 : 
4170 (1967). (c) C. Allen Bush and H. A. Scheraga. 
Biochemistry 6^: 3036 (1967). (d) Y. Inoue, S. Aoyagi and 

K. Nakanishi. Tetrahedron Letters, 3575 (1967). 

8. (a) R. C. Ferguson and W. D. Phillips. Science 157 : 257 
(1967). (b) C. C. McDonald, W. D. Phillips, and J. Lazar. 

J. Am. Chem. Soc. 89: 4166 (1967). (c) C. C. McDonald, W. D. 
Phillips, and J. Penswick. Biopolymers 3_' (1965). 
(d) K. H. Scheit, F. Cramer, and A. Franke. Biochim. et 
Biophys. Acta 145: 21 (1967). (e) G. K. Helmkamp and N. S. 
Kondo. ibid. 145: 27 (1967). (f) Y. Inoue and S. Inoue. 
Biochem. Biophys. Res. Commun. 28_i 973 (1967). 

9. (a) E. J. Gabbay.. Biopolymers 5.: 727 (1967). (b) M. M. Fishman, 
T. Isaac et al. Biochem. Biophys. Res. Commun. 29: 378 (1967). 

(c) Y. Ohba and Fromageot. Europ. J. Biochem. 1^: 147 (1967). 



- 10 - 



10. (a) F. Pochon and A. M. Mlchelson. Biochim. Biophys. Acta 
149 : 99 (1967). (b) A. M. Michelson and C. Monny. ibid . 
149: 107 (1967). (c) F. Pochon and A. M. Michelson. ibid . 
145 : 321 (1967). (d) A. M. Michelson and F. Pochon. ibid . 
114 : 469 (1968). (e) J. Massoulie, A. M. Michelson, and 

F. Pochon. ibid. 114_: 16 (1966). (f) F. Pochon and A. M. 
Michelson. Proc. Natl. Acad. Sci. 53: 1425 (1965). (g) 
J. Brahams, A. M. Michelson, and K. E. VanHolde. J. Mol. 
Biol. 15: 467 (1966). 

11. (a) J. T. 0. Kirk. J. Mol. Biol. 28: 171 (1967). (b) . 

R. L. C. Brimacombe and C. B. Reese. J. Mol. Biol. 18: 529 

(1966) . (c) D. B. Ludlem. Mol. Pharmacol. 1'. 585 (1966). 
(d) D. B. Ludlem. Biochim. Biophys. Acta 119^: 630 (1966). 

12. (a) B. Singer and H. Fraenkel-Conrat. Proc. Natl. Acad. Sci. 
58_: 234 (1967). (b) H. G. Wittmann and B. Wittmann-Liebold . 
Cold Spring Harbor Symp. Quant. Biol. 31: 527 (1966). (c) 

A. Siegel. Advances in Virus Research IJ: 25 (1965). 

13. J. T. Marvel and A. J. Jaworski, unpublished results. 

14. A. Holy and K. H. Scheit. Biochim. Biophys. Acta 138^: 230 

(1967) . 



TEMPLATES IN A TEST TUBE: ENZYMATIC REPLICATION OF DNA 

Sigmund Schwimmer 
Western Utilization Research and Development Division, USDA 

Albany, California 

Topics that illustrate accomplishments in the field of 
enzymatic replication of DNA and its possible role in agriculture 
are: the discovery and isolation of DNA polymerase; the role of 
this enzyme in the putative "creation of life in a test tube"; 
and studies carried out in the laboratory of Dr. James Bonner at 
California Institute of Technology on the mode, enzymes, template 
(nucleohistone) , and herbicidal inhibition of DNA synthesis in 
the cells of higher plants. 

Although the elucidation of the structure of DNA by Watson 
and Crick in 1953 (1) led to a logically deducible model for gene 



- 11 - 



replication, there was no enzymological or experimentally proved 
basis for this model until the discovery and purification of 
Escherichia coll DNA polymerase in 1958 (2). The chemical 
mechanism was shown to involve a nucleophilic attack of the 
3'-0H of the last deoxyribose of a growing polydeoxynucleotide 
on incoming deoxynucleoside triphosphate to form a phospho- 
diester bond with the liberation of pyrophosphate. The require- 
ments of the reaction and most of the properties of the newly 
synthesized DNA fulfilled most of the requirements for a true 
replication from DNA template. However, in contrast to the 
Watson-Crick model and in vivo observations, the poljnnerase did 
not catalyze synthesis of complementary strands of DNA from the 
same end of a linear double-stranded DNA template (Y-type 
replication) . This resulted in a highly branched DNA product 
which was undenaturable and had unusual electrophoretic properties 
(4). Some of the DNA synthesized at high template concentrations 
appeared to be single-stranded (5). Furthermore, the product DNA 
did not possess biological activity. These observations suggested 
that the polymerase was a "repair" enzyme rather than a true 
replicase. This problem was circumvented by the use as template 
of single-stranded circular DNA of the bacterial virus (})X 174. 
With the aid of auxiliary enzymes (including the newly discovered 
DNA "joining enzyme" (6)) it was possible to synthesize and iso- 
late in a test tube, the infectious chromosome of the bacteriophage 
with its complete set of genes from nonliving chemicals (7). 

The Watson-Crick model of replication predicts that the DNA 
of the first generation of daughter cells in a growing culture of 
cells will consist of a hybrid containing parent and newly synthe- 
sized DNA (semiconservative mode of replication). By growing 
tobacco cells in an N-15-contalning medium and then transferring 
to an N-14-containing medium and determining the sedimentation 
characteristics of the isolated DNA therefrom, it was shown that 
the mode of replication of DNA in higher plants (like that of 
bacteria and animals) , is semiconservative (8) . 

After several false leads (9) , DNA polymerase activity was 
demonstrated for the first time in plant tissue, the roots of the 
mung bean seedling (10). Although the specific activity of crude 
extracts is about 0.1 percent that of bacteria, the activity of 
DNA polymerase per unit weight of DNA is the same for both plants 
and bacteria. This illustrates the general principle that a cell 
will make just enough enzyme in response to its requirement for 
substrate or product. 

The natural template for DNA synthesis in plants is the DNA 
contained in the chromosomes of the nucleus of the plant cell. The 
chromosomes of the pea embryo can be prepared and isolated as a 
preparation of chromatin which consists largely of DNA partially 
complexed to basic proteins known as histones (11). Chromatin, 



- 12 - 



because of its free DNA, can serve as template for messenger RNA 
synthesis, which in turn directs enzyme (protein) synthesis. 
However, the fraction of chromatin DNA covered with histone 
(nucleohistone) cannot serve as template for RNA synthesis . Thus 
histone plays a role in the regulation of the gene expression by 
repressing those genes not needed at a given time in the life 
cycle of the plant. Nucleohistones can serve however, as template 
for DNA synthesis by DNA poljnnerase (12). Furthermore reconsti- 
tuted nucleohistones, prepared by combining free DNA with 
various histone fractions, can also support DNA synthesis (13). 

From studies on polymerase kinetics (lA), susceptibility 
of product DNA to certain nucleases (15) and from ultracentrif ugal 
and electrophoretic properties of the newly synthesized DNA (16) , 
it is concluded that plant nucleohistone supports limited 
synthesis of double-stranded, unbranched DNA, that most of the 
DNA product is complexed with the template (but not in the same 
manner as the DNA of the template is complexed with histone) , 
and that there is very little transfer of histone from template 
to product. These data are in agreement with the view that 
histones are conserved during cell division. 

The herbicide 2 , 4-dichloroghenoxyacetic acid inhibits the 
activity of DNA polymerase at 10 M (17). This inhibition may 
contribute to the toxic effect of this herbicide, by preventing 
or slowing down cell division. 

Literature Cited 



1. 


J. 


D. Watson and F. H. C. Crick. Nature 171: 737 (1953); 




Cold Spring Harbor Symp. Quant. Biol. 18^: 123 (1953). 


2. 


I. 


R. Lehman, M. J. Bessman, E. S. Simms, and A. Kornberg. 




J. 


Biol. Chem. 233: 163 (1958). 


3. 


C. 


L. Schildkraut, C. C. Richardson, and A. Kornberg. 




J. 


Mol. Biol. 9: 24 (1964). 


4. 


S. 


Schwimmer and B. M. Olivera. J. Mol. Biol. 20: 585 (1966). 


5. 


S. 


Schwiramer. Life Sci. 5^^ 1^15 (1966). 


6. 


B. 


M. Olivera and I. R. Lehman. Proc. Natl. Acad. Sci. 57: 




1426 (1967). 


7. 


M. 


Goulian, A. Kornberg, and R. L. Sinsheimer. Proc. Natl. 




Acad. Sci. 58: 2321 (1967). 


8. 


P. 


Filner. Exptl. Cell Res. 39: 33 (1965). 



- 13 - 



9. S. Schwiramer, S. Kabat, and P. Filner. Biochim. Biophys. 
Acta 108: 150 (1965) . 



10. S. Schwimmer. Phytochemistry 5^: 791 (1966). 

11. R. C. Huang and J. Bonner. Proc. Natl. Acad. Sci. 48: 1216 
(1962). 

12. S. Schwimmer and J. Bonner. Biochim. Biophys. Acta 108 : 67 
(1965). 

13. S. Schwimmer. Life Sci. 1247 (1965). 

14. S. Schwimmer. Fed. Proc. _27_: 804 (1968). 

15. S. Schwimmer and A. Aronson. Biochim. Biophys. Acta 134 : 
59 (1967). 

16. S. Schwimmer and B. M. Olivera. Biopolymers 4^: 953 (1966). 

17. S. Schwimmer. Plant Physiol. 43_: 1008 (1968). 



RNA TRANSCRIPTION 

J. S. Krakow 
Space Sciences Laboratory 
University of California, Berkeley 

The transcription of RNA from the DNA template is catalyzed 
by RNA poljmierase. This enzyme was first demonstrated in rat liver 
nuclei by S. B. Weiss (1) and subsequently extensively purified 
from several bacterial species including E. coli (2), M. lysodeik - 
ticus (3) , and A. vinelandii (4) . The in vitro characteristics 
of RNA polymerase are similar ;_|_|^or RNA synthesis RNA pol5mierase 
requires a divalent cation (Mg , Mn"*"^) , ribonucleoside triphos- 
phates (ATP, UTP, OTP, CTP) and a template (5). The template in 
vitro is not limited to native DNA, since single-stranded DNA and 
polyribonucleotides will also direct polynucleotide synthesis (6). 
In addition to template-directed reactions RNA pol37merase will 
also catalyze two unprimed reactions leading to poly A: poly U 
homopolymers (7) and rIC copoljrmer (8). The unprimed reactions 



- 14 - 



demonstrate the complementary nature of the RNA polymerase reac- 
tion, since the products obey the Watson-Crick rule of hydrogen 
bonding between A and U (or T) , and I (or G) and C. This 
complementarity, by virtue of the specificity elicited by 
hydrogen bonding, is seen for all RNA (and DNA) polymerase 
reactions. RNA synthesized in the DNA-directed reaction is 
complementary in composition and sequence to that of the template; 
this has been shown by determination of base composition, nearest 
neighbor frequencies (5) and hybridization (9) . 

RNA synthesis in vivo is known to be as3nraiietric ; that is, 
within each operon only one strand of the DNA duplex is trans- 
cribed into RNA. This is due to the initial event in RNA 
synthesis; RNA polymerase binds to DNA at specific sites, 
resulting in a DNA-enzjmie complex. From the work of Szybalski 
and coworkers (10) it would appear that these binding sites 
consist of pyrimidine-r ich tracts on the DNA molecule. In an 
extreme case, that of T7DNA, these tracts are dC-rich and are 
all in one strand of the DNA duplex. Therefore it is this strand 
which is transcribed into RNA. Szybalski has suggested that the 
dC- and dT-rich regions represent a part of each operator region. 
Since the represser binds to the operator region, the presence 
of represser molecules would prevent formation of the DNA-enzyme 
complex and thereby block transcription of that operon. The DNA- 
enzyme complex can be demonstrated by sucrose density gradient 
centr if ugation (11) and by retention of the complex on cellulose 
nitrate filters (12). 

Initiation of RNA synthesis occurs by incorporation of an 
intact ribonucleoside triphosphate to form the 5' end of the RNA 
chain. This is the first nucleotide incorporated and chain elonga- 
tion occurs in a 5 ' 3 ' direction (13). Initiation occurs 
predominantly by incorporation of ATP and GTP and only to a small 
extent (less than 5 percent) by CTP or UTP (13,14). The GTP/ATP 
ratio varies with the DNA template used and may reflect the ratio 
of dC- and dT-rich promoter regions to the RNA polymerase binding 
sites on the DNA. RNA polymerase binds to the promoter site on 
the DNA and causes a localized melting of the helix. As poly- 
nucleotide synthesis progresses the RNA is found in the form of a 
transient RNA:DNA hybrid. As polymerase moves along the template 
the DNA helix reforms and displaces the RNA from this hybrid 
intermediate . 

RNA synthesis in vitro does not show linear kinetics and 
after 60 minutes at 37° the reaction plateaus and no further RNA 
is formed. This has been shown to be due to product inhibition 
whereby the RNA synthesized presumably "clogs" the template site 
of polymerase. It is possible to obtain nearly linear kinetics 



- 15 - 



by running the reaction in the presence of ribonuclease to 
hydrolyze the RNA as it is formed and using PP. released as a 
measure of poljrmerase activity (15) . 

It is an accepted fact that in vivo synthesis of protein 
and RNA are coupled. Stent (16) has suggested a model whereby 
translation and transcription are coupled by the movement of the 
ribosomes across the messenger RNA; this "strips" the RNA off the 
polymerase and prevents product inhibition from occurring. Such 
a system can be set up _in vitro and it has been shown that the 
addition of ribosomes competent in binding RNA will stimulate RNA 
poljmierase (17). 

Literature Cited 

1. S. B. Weiss. Proc. Natl. Acad. Sci. 46: 1020 (1960). 

2. M. J. Chamberlin and P. Berg. Proc. Natl. Acad. Sci. 48_: 81 

(1962) . 

3. T. Nakamoto, C. F. Fox, and S. B. Weiss. J. Biol. Chem. 239 : 
167 (1964). 

4. J. S. Krakow and S. Ochoa. Biochem. Z. 338: 796 (1963). 

5. J. Hurwitz and J. T. August. Progr . Nucl. Acid Res. _1: 59 

(1963) . 

6. J. S. Krakow and S. Ochoa. Proc. Natl. Acad. Sci. _49: 88 
(1963). 



7. D. A. Smith, R. L. Ratliff, D. L. Williams, and A. M. Martinez. 
J. Biol. Chem. 242: 590 (1967). 



8. J. S. Krakow and M. Karstadt. Proc. Natl. Acad. Sci. 58: 
2094 (1967). 

9. E. P. Geiduschek, T. Nakamoto, and S. B. Weiss. Proc. Natl. 
Acad. Sci. 47_: 1405 (1961). 

10. W. Szybalski, H. Kubinsky, and P. Sheldrick. Cold Spring 
Harbor Symp. Quant. Biol. 3±: 123 (1966). 

11. J. P. Richardson. J. Mol. Biol. 21: 83 (1966). 

12. 0. W. Jones and P. Berg. J. Mol. Biol. _22: 796 (1966). 

13. U. Maitra and J. Hurwitz. Proc. Natl. Acad. Sci. _54: 815 
(1965). 



- 16 - 



lA. J. S. Krakow and W. J. Horsley. J. Biol. Chem. 242: 4796 
(1967) . 

15. J. S. Krakow. J. Biol. Chem. 241: 1830 (1966). 



16. G. S. Stent, in. Organizational Biosynthesis (ed. Vogel et al.) 
(1967) , p. 99~ 

17. M. Revel and F. Gros. Biochim. Biophys. Res. Commun. 2_7: 12 
(1967). 



REGULATORY MECHANISMS INVOLVING ENZYME FUNCTION, 
SYNTHESIS AND DEGRADATION 

Philip Filner 
MSU/AEC Plant Research Laboratory 
Michigan State University, East Lansing 

Since the essence of progress in agriculture is learning 
how to maximize desirable and minimize undesirable biological 
characteristics, it is important to understand the natural mech- 
anisms by which the characteristics are regulated. All biological 
characteristics are the result of the combination of enzyme- 
catalyzed reactions, and so it is appropriate to focus attention 
on the mechanisms by which catalysis is regulated. There are 
thirteen points between the gene and the enzyme-catalyzed reaction 
at which specific control of the reaction rate can be exercised 
(fig. 1). 

The work of Bonner, Huang, and Gilden (1) indicated that the 
synthesis of pea-seed globulin is controlled by the accessibility 
of the DNA region in which the amino-acid sequence of pea-seed glob- 
ulin is encoded. Chromosomal material isolated from the globulin- 
synthesizing tissue, the cotyledon, could serve as template for the 
synthesis of m-RNA, which in turn could be translated in pea-seed 
globulin, while a nonsynthesizing tissue, the pea bud, yielded 
chromosomal material with far less, if any, of this activity. De- 
proteinized DNA from either source served equally well as template 
for synthesis of pea-seed globulin messenger. 

Experiments of Leaver and Key (2) have shown that the increase 
in protein synthesis seen in aged carrot root discs is accompanied 
by the formation of polyribosomes, which have greater amino acid 
incorporating activity in_ vitro . The formation of these polyribo- 
somes was dependent upon the synthesis of an RNA which could be 
inhibited by actinomycin D, presumably an m-RNA. These results 



- 17 - 



CATALYSIS 

SUBSTRATE, SUBSTRATE-^I^^^ACCESSIBLE ,^ INACCESSIBLE 

COFACTOR ~^ -^COFACTOR ENZYME '^^'^) ENZYME 

o ■ 

INACTIVE ACTIVE 
ENZYME ^ J;' ENZYME 

O 

UNSTABLE , . STABLE 

ENZYME ^^X^'^ENZYME 

UNFINISHED - FINISHED 

ENZYME "j^^^^NZYME 



NO PROS- ' 

PROS- , .TTHETIC 

THETIC ^ -^JROUP 
GROUP 



iSEC. 1 fsEC. ^ 

TERT. y STRUC--^7)— NO JtERT. > STRUC- 
QUART. TURE | QUART.] TURE 



NO POLYPEPTIDE (6)-^^ POLYPEPTIDE 



NO POLYSOME— (4)*-P0LYS0ME^^ 



I _ I^^^AMINOACY 



/ 



,„ _ • „„. -AMINOACYL NO AMINOACYL 

NO m-RNA ^— (2)-»-m-RNA „„. (5>— . 

t-RNA ' ^ t-RNA 




INACCESSIBLE ,|S_ACCESSIBLE 

DNA REGION ^ ^*T3NA REGION 



t-RNA -*<3) NO t-ENA 



Figure 1. Points of control of reaction rate. 

indicate that step 2 is an important regulatory point. The work of 
Marcus and Feeley (3), however, has indicated that synthesis of 
m-RNA is not necessary for the initial increase in protein synthesis 
during wheat embryo imbibition. They were able to activate the 
protein-synthesizing capacity of extracts of unimbibed embryos, the 
result being polyribosome formation. In this system, step 3 is the 
control point. 

The importance of t-RNA as a potential point of specific 
regulation is discussed by Doi elsewhere in this report, based on 
his work on t-RNA' s of sporulating B. subtilis . 



When a general stimulation of protein synthesis occurs, as 
in the aged tissue disc, or the imbibed embryo, the question arises: 
Is the stimulation specific for any proteins, or are all proteins 
synthesized more rapidly? Patterson and Trawavas (4) have recently 
reported data that indicate that indole acetic acid induces the 
synthesis of specific proteins in pea-stem segments. 



- 18 - 



The only case in higher plants where specific induced 
enzyme activities have been shown to be synthesized de^ novo are 
the a-amylase and protease induced by gibberellic acid in barley 
aleurone (5,6,7,8). 

Enzyme degradation or stabilization (regulatory step 10) 
has received attention by a number of people who have studied the 
control of mammalian liver enzymes. Tryptophan pyrrolase, for 
instance, increases in level in rat liver in response to trypto- 
phan injections because tryptophan stabilizes the enzyme without 
altering the rate of synthesis (9). The level of tyrosine trans- 
aminase in rat liver is unaffected by cycloheximide because this 
inhibitor blocks both its synthesis and its degradation (10). 
Prednisolone induces an increase in alanine transaminase in rat 
liver by accelerating both synthesis and degradation, but synthe- 
sis is accelerated relatively more (11) c 

An example of the use of regulatory step 11 is in the work 
of Pressey (12), who has isolated and purified a protein from 
potato tubers which specifically inhibits invertase. This is 
particularly noteworthy, since a number of workers are studying 
the invertase activity which develops during aging of storage 
tissue discs (13,14). 

The RNAse induced by gibberellic acid in barley aleurone 
cells (15) is accumulated in the cells and is later secreted. 
Thus we see that step 12 is also used to regulate catalytic 
activity. 

One case in which the study of enzyme regulation has result- 
ed in some suggestive leads for future developments in agriculture 
is the effect of the herbicide simazine on nitrate reductase (16). 
Subtoxic levels of simazine cause an increase in nitrate reductase. 
Plants growing on nitrate plus simazine accumulate more protein, 
up to 80 percent more, than plants on the same level of nitrate 
(17). On the other hand, simazine has no effect on the protein 
content of plants growing on ammonia. Recent work has shown that 
simazine also increases nitrate and asparagine accumulation (18). 
It appears from these results that the activities of nitrate 
permease and nitrate reductase are the limiting factors in protein 
synthesis on low levels of nitrate, and that simazine increases 
the rates of these two enzyme-catalyzed processes in a manner 
which is not yet understood, but which has the desirable effect of 
increasing the protein content and hopefully the nutritional value 
of the plant. 



- 19 - 



Literature Cited 

1. J. Bonner, R.-C. Huang, and R. V. Gilden. Proc. Natl. Acad. 
Sci. U.S. 50: 893 (1963). 

2. C. J. Leaver and J. L. Key. Proc. Natl. Acad. Sci. U.S. 57_: 
1338 (1967). 

3. A. Marcus and J. Feeley. Proc. Natl. Acad. Sci. U.S. 56 : 
1770 (1966). 

4. B. D. Patterson and A. J. Trawavas. Plant Physiol. 4^: 1081 
(1967). 

5. J. E. Varner and G. R. Chandra. Proc. Natl. Acad. Sci. U.S. 
52: 100 (1964). 

6. J. E. Varner. Plant Physiol. 39.: 413 (1964). 

7. P. Filner and J. E. Varner. Proc. Natl. Acad. Sci. U.S. 58: 
1520 (1967). 

8. J. V. Jacobsen and J. E. Varner. Plant Physiol. 4_2: 1596 
(1967). 

9. R. T. Schimke. J. Biol. Chem. 239: 3808 (1964). 



10. F. T. Kenney. Science 156: 525 (1967). 

11. H. L. Segal and Y. S. Kim. Proc. Natl. Acad. Sci. U.S. 50: 
912 (1963). 

12. R. Pressey. Plant Physiol. _42: 1780 (1967). 

13. P. Kaufman, N. Ghosheh, and H. Ikuma. Plant Physiol. 43_: 29 
(1968). 

14. J. D. Bacon, I. R. MacDonald, and A. H. Knight. Biochem.. J. 
94: 175 (1965). 

15. M. Chrispeels and J. E. Varner. Plant Physiol. 42: 398 (1967). 

16. J. A. Tweedy and S. K. Ries. Plant Physiol. 42: 280 (1967). 

17. S. K. Ries, H. Chmiel, D. R. Dilley, and P. Filner. Proc. 
Natl. Acad. Sci. U.S. 58: 525 (1967). 

18. S. K. Ries, unpublished results. 

- 20 - 



CELLULAR ORGANIZATION 



E. H. Mercer 
University of California 
Medical Center, San Francisco 

In intact organisms the biochemical reactions we have been 
discussing take place in cells, which subdivide all known kinds 
of living matter into small closed compartments. With the 
exception of bacterial cells, all other cells are further compart- 
mentalized to provide smaller reaction chambers, where reactants 
and enzjmies meet. The walls of these chambers are thin membranes 
whose predominantly lipid character renders them relatively 
impervious to aqueous solutions and hydrophilic molecules. The 
cell's aqueous phase is thus divided into volumes having a limited 
and controlled intercommunication. Biological membranes also 
have a protein moiety, probably enzymatic, and on this constituent 
depends the second important property of membranes, their selective 
permeability, which creates within their enclosures a special micro- 
environment by controlling the ingress and egress of metabolites 
and ions . 

It is apparent then, that viewed as a chemical machine, the 
functioning of a cell is not determined solely by the enzymes coded 
in its genome. Enzjnnes and substrates are not just lumped together. 
Structural segregation plays a role and structure is largely a 
matter of membrane topology. 

Cells are very small (mostly 20-30 y in diameter) and can be 
studied only with the aid of microscopes. Their internal details 
in fact can only be visualized effectively by means of the electron 
microscope. The great post-war renaissance of morphology stems 
from the perfection of this instrument and of methods of processing 
biological materials for it. 

All cells have an external or plasma membrane (fig. 1, PM) 
which defines their limits, holds their bits and pieces together 
and is the first outpost of the physiological system creating and 
maintaining the special internal environment. In addition most 
large cells have an inner chamber or nucleus in which, except at 
division, the genetic apparatus is segregated from the rest of 
the cell (fig. IB). Bacteria, the smallest and simplest of cells, 
lack the nuclear compartment (fig. lA) . 

In the cytoplasm of nucleated cells are other small membrane- 
bounded organelles having special functions: mitochondria and in 
plants chloroplasts and other plastids. Since other speakers will 
discuss these organelles, they may be passed over here. In any 
case, fortunately, they behave as almost independent units. 



- 21 - 




Figure 1. The primary classification of cells: (A) The 
protokaryote or one-envelope cells. The genetic apparatus 
lies within the cytoplasm and is not enclosed by a second 
nuclear membrane (bacteria and blue-green algae) . (B) The 
eukaryote (two envelope cells) . The genetic apparatus is 
segregated from the cytoplasm by a double-layered nuclear 
membrane (NM) . PM is the plasma membrane 
and W is the cell wall. 

Other important constituents, which also can only be 
mentioned in passing, are: microtubules , an intracellular skeletal 
component used extensively to stiffen the cytoplasm where needed 
and to build the mitotic apparatus, the shafts of cilia, sperms, 
and other elongated protrusions; and centrioles , small, still 
mysterious objects with the property of organizing microtubules. 

Most cellular activities are mediated by biochemical reac- 
tions catalyzed by enzymes. A more or less extensively developed 
system of membranes is devoted to the task of collecting enzymes, 
channeling them into special compartments, bringing metabolites 
to these enclosures, and managing the products of their action. 
This membrane system is the endoplasmic reticulum (ER) . So 
important is this organelle that we are able to describe much of 
the cell's total activity in terms of its many manifestations. 
The first steps in protein synthesis up to polypeptide assembly 
on ribosomes (transcription and translation) are the same in all 
systems. Membranes play a small role here. But from the moment 
a polypeptide leaves a ribosome, its fate is in the hands of the 
ER. 

The ER divides the cell space into two parts: inside the 
closed membranous system and outside (fig. 2) . Ribosomes are 
(with the nucleus and the genetic molecules) outside the reticulum 
and a fundamental branching of the flow of newly synthesized 
proteins is established by the dividing membrane itself. 



- 22 - 



Figure 2. The two pathways of protein synthesis: I and II. 
Beginning each pathway a nucleotide sequence of the nuclear 
DNA corresponding to a gene sheds a messenger RNA (mRNA) 
which passes out of the nucleus, probably via a pore in the 
nuclear membrane NM, into the cytoplasm. In the cytoplasm 
the mRNA (according to its code) will either: (I) pass to 
an element of the rough ER where, associated with a membrane- 
bound ribosome, it assembles a polypeptide which will be col- 
lected within the sac of the ER and be isolated from the cell 
sap; or (II) pass to a free ribosome where the polypeptide 
assembled will be shed freely into the cytoplasm and remain 
the cell sap. PM = plasma membrane, NM = nuclear membrane, 
R = ribosomes, M = messenger RNA, p = polypeptide, P = protein. 

Polypeptides, synthesized on ribosomes attached to the membrane, 
pass through it and are collected in closed sacs of the ER (fig. 
2 I) ; those formed on "free" ribosomes pass directly into the 
cell sap outside the membrane system (fig. 2 II). We thus dis- 
tinguish "free proteins" and "membrane enclosed proteins." 

In multicellular organisms cells are specialists, which 
means they do one main job and they do this by synthesizing one 
(or a few) enzymes or structural proteins. Such cells are said 
to be differentiated and their cytology reflects this fact. In 
some, free ribosomes predominate and the proteins collect in the 
cell sap to form specialized structures or accumulations on which 
the differentiated function depends. For example the haemoglobin 
in red cells converts them into oxygen transports; the fibrous 
proteins in muscle cells are the basis of contractility; the 
toughened proteins of epidermal cells form a protective coat. 
In ether cells a variety of functions are based on proteins 



originating from bound ribosomes and therefore enclosed in branches 
or vacuoles of the ER. These activities (summarized in fig. 3) 
usually involve the participation of an important organelle, the 
Golgi apparatus, which may be regarded as a specialized region of 
the ER not associated with ribosomes (fig. 3 at G). 



Figure 3. Intracellular mtovements and Golgi involvement of 
proteins synthesized on the rough surfaced ER. Note: The 
diagram is a composite of several pathways not all seen in 
one cell. Each may be elaborated and become the dominant 
structural and functional feature of a differentiated cell. 
In each case the protein is collected from the ribosomes in 
a sac of the ER from where, according to its composition and 
role, it may follow any of the several courses: (1) Extra- 
cellular secretion of enzymes, etc. The protein is transferred 
by small vesicles to condensing vacuoles (CV) derived from the 
Golgi (G) and is passed ultimately through the plasma membrane 
PM at E. (2 and 3) Enzymes polymerizing polysaccharides move 
to Golgi vesicles (M and N) where the monomers enter and are 
pol5mierized . In (2) polysaccharide only is formed; in (3) 
polysaccharides are linked to a protein to form mucopoly- 
saccharides (mucins). (4 and 5) Hydrolytic, degradative 
enzymes collect in Golgi vesicles to fom lysosomes L. These 
may be used either (4) to digest the contents of food vacuoles 
(P) formed by phago- or pinocytosis or (5) to digest damaged 
parts of the cell itself which have been walled off in an 
autophagosome A. (6) Various synthetic enzymes may find their 
substrates collected in vesicular or tubular extensions of the 




PM 



ER (V) e.g., steroids and lipids synthesis. 



- 24 - 



Cells, synthesizing proteins for export, e.g. the pancreatic 
acinar cells, pass their enzymes from the ER sacs in small vesicles 
to larger vesicles shed from the Golgi (fig. 3 at 1) where they are 
condensed to granules. Since these are still enclosed in membranes 
they can escape from the cell by the fusion of the vacuole membrane 
with the plasma membrane as at figure 3, E. Very similar events 
occur with enzymes which polymerize polysaccharides or mucins 
(polysaccharides plus protein). These are enclosed in Golgi vesicle 
where they polymerize the carbohydrate monomers transported there 
by membrane activity. The products are secreted as before (fig. 3 
at 3). Thus in secretory cells in general we commonly find an 
abundance of ribosome-covered membranes and an elaborate Golgi 
apparatus . 

Comparative cytological studies have shown that, like the 
basic biochemistry itself, these intracellular patterns are common 
to all organisms — with the exception of bacteria which are too 
small to contain them. In plants protein secretion is uncommon 
and therefore the ribosome covered ER is scanty. Polysaccharide 
secretion to form cell wall in contrast is highly developed and 
is associated with an abundant Golgi apparatus and vesicles 
containing the poljmierized enzymes (pathway 2 and 3 of fig. 3). 

An important function in cells is the digestion of food or 
cellular debris. Food particles enter in vacuoles as at (4) in 
figure 3 (phagocytosis), and the degradative enzjmies, previously 
bagged up in a vacuole called a lysosome, derived as before from 
the ER and Golgi, are added to this vacuole to digest its contents. 
Damaged cellular organelles are bagged up by membranes to form 
auto-phagocy tic vacuoles and are dealt with similarly (fig. 3 at 5) 
Other enzjmies, funneled into tubular branches of the ER, are 
responsible for lipid and steroid synthesis in cells producing 
these (fig. 3 at 6). Some enzymes, which serve to transport water 
or salts across membranes, may find their way to and be adsorbed 
on extensive meandering, smooth-surfaced tubules, or pleated 
membranous folds connected with the plasma membrane. Examples 
are found in kidney cells and in the chloride cells of fish gills. 

As mentioned above, differentiated cells elaborate, prefer- 
entially, one or another of these functions and the membranous 
system appropriate to it. The several possibilities, summarized 
in figure 3, may each individually be developed, with the suppres- 
sion of other pathways, to become the dominant membranous pattern 
of a differentiated cell. The characteristic cytological patterns, 
long familiar at the histological level, thus find their origins 
and function in the synthesis and transport by their internal 
membrane system of their most fundamental constituents, enzymes 
and proteins. 



- 25 - 



Further Reading 

The Cell. Vols. 1-6 (eds, J. Brachet and A. E. Mir sky) , 
Academic Press, New York (1961). 

The Cell. Its Organelles and Inclusions. D. W. Fawcett and 
W. B. Saunders. Philadelphia (1966). 

The Structure and Functions of Cells, 2nd Ed. E. H. Mercer. 

Natural History Press and Doubleday, New York (1967). 

The Cell. C. P. Swanson. Prentice Hall, New Jersey (1960). 

The Living Cell. Scientific American 205_ (1961). 



Cell Structure and Function. A. Loewy and P. Siekevitz. 
Holt, Rinehart and Winston, New York (1965). 



CHLOROPLAST STRUCTURE AND DEVELOPMENT 

W. M. Laetsch 
Department of Botany 
University of California, Berkeley ^ 

Chloroplasts are particularly good candidates to provide a 
focal point for molecular biology and agriculture. They are self- 
replicating subcellular units which can be centrifuged, 
homogenized, and incubated with all the mixtures beloved by 
molecular biologists. They appeal to the agriculturalists because 
increased world food production must ultimately depend upon 
improving photosynthetic efficiency. This depends upon knowledge 
of their structure and function. 

This paper is concerned with two topics. The first is a 
discussion of differentiation and senescence in tobacco chloro- 
plasts, and the second revolves around chloroplast specialization 
in plant groups with unique features of photosynthesis. 

The "typical" chloroplast is a disc-shaped organelle about 
5-8 y in diameter. The outer envelope is a double membrane, and 
a complex series of internal membranes floats in a matrix called 



- 26 - 



the stroma. The enzymes involved in the dark reaction of photo- 
synthesis are located in this membrane-free fraction. Chlorophyll 
in chloroplasts is localized within the internal membranes. This 
system consists of flattened vesicles which are either arranged 
in stacks called grana, or occur singly and connect the grana. 
Storage products such as starch, lipid, or protein are found in 
the stroma. 

Chloroplasts are considered to be self -replicating organ- 
elles and schemes for their "life cycles" have been proposed 
(1,2). Developmental sequences can be traced from organelles in 
the shoot apex to mature chloroplasts in the leaves. The pro- 
plastids in the apex have a double membrane, are 1-3 y in diameter, 
and possess some stroma lamellae. In addition, they frequently 
have prolamellar bodies. These are paracrystalline arrays of 
tubules. In tobacco proplastids, we have also observed single- 
membrane bound bodies with an electron dense interior. These 
structures are frequently attached to prolamellar bodies. 

The development of the proplastid into a chloroplast 
parallels the development of the leaf. Both proplastids and 
partially mature chloroplasts divide. The development is charac- 
terized by growth of the plastid and by the differentiation of 
the internal membrane system. Grana appear both in association 
with the prolamellar body and with lamellae produced from the 
inner limiting membrane. In tobacco, grana are also associated 
with the single-membrane bound body described above. As 
development proceeds, this body becomes granular and eventually 
the contents consist of fibrils the size of those shown to be 
DNA (1). Remnants of the prolamellar body persist until the 
last stages of tobacco chloroplast development. 

Most studies of chloroplast development have utilized 
dark-grown material which is subjected to light. The organelles 
in dark-grown leaves which develop into chloroplasts are now 
frequently called etioplasts. They are usually larger than the 
proplastids of the stem apex and they usually contain prominent 
prolamellar bodies. Tobacco etioplasts also contain the single- 
membrane bodies found in proplastids in light-grown plants. 
When dark-grown leaves are exposed to the light, the development 
of chloroplasts is quite rapid. The prolamellar bodies disappear 
and grana develop. There is a general impression that the 
prolamellar body is always intimately involved with the formation 
of grana. This is not always the case, since perfectly good 
chloroplasts can develop in tobacco tissue cultures even though 
the etioplasts in dark-grown cultured tissue do not have 
prolamellar bodies (Laetsch and Stetler, 1965). At present there 
is a general feeling that the earlier schemes for chloroplast 



- 27 - 



development are too rigid and do not give recognition to the 
evidence that there are many ways to make a chloroplast. 

Most studies of chloroplast development have stopped with 
the mature chloroplast in spite of the fact that senescence is a 
natural part of the developmental sequence. When a leaf senesces 
on the plant, the most obvious change is bleaching, of the green 
pigment. The initial changes in the chloroplast are the degra- 
dation of stroma lamellae. This is followed by increased density 
and loss of membrane detail in the grana. When tobacco leaves 
have lost 80 percent of their chlorophyll, the chloroplasts have 
lost their stroma lamellae and the grana stacks are recognizable 
as electron-dense clusters. Lipid globules frequently increase 
during senescence. This natural senescence can be contrasted 
with a controlled senescence in cultured leaf tissue. Sterile 
leaf discs of tobacco can be kept alive for many weeks in the 
dark on a simple mineral-salts-and-sucrose medium. Chlorophyll 
is lost, but the morphology of the chloroplasts is quite differ- 
ent from those discussed above. The grana disappear, but the 
stroma lamellae persist. When almost all the chlorophyll is 
gone, the chloroplasts are without grana. If the leaf discs 
are transferred at this stage to a medium promoting cell division, 
the chloroplasts revert to the proplastid condition. Since this 
tissue will turn green when exposed to light, it is possible to 
carry a chloroplast through a complete cycle of dedif f erentiation 
and rediff erentiation. 

The previous description of chloroplast development is 
more or less typical of a variety of plants like tobacco and bean, 
but it cannot be said that all chloroplasts are alike. Tropical 
grasses, such as sugar cane, are a case in point. They have two 
types of chloroplasts and these dimorphic chloroplasts occur in 
separate cell layers. The bundle sheath cells surrounding the 
vascular bundles possess large plastids which do not have grana 
and which store a lot of starch. The mesophyll cell layer is 
external to the bundle sheath cell, and it contains "typical" 
chloroplasts which store very little starch. Some grasses in 
this general group, such as corn, do have bundle-sheath-cell 
chloroplasts with rudimentary grana. These dimorphic chloro- 
plasts are of special interest because the grasses possessing 
them also have a pathway of photosynthetic CO^ fixation which 
differs from the Calvin cycle. The primary fixation products in 
these grasses are C^-dicarboxylic acids (5). These grasses 
possess other features in common. They are resistant to photo- 
saturation (2,3); they fail to leak respiratory CO2 during the 
light period (4); and they have low CO2 compensation values (8). 
In addition. Dr. Bruce Smith (Division of Geology, California 
Institute of Technology) has shown that they have much higher 
13^,12^ 

C/ C ratios than temperate grasses. 



- 28 - 



The development of these dimorphic chloroplasts Is quite 
interesting. The proplastids in the meristem at the base of the 
leaf are very similar even though the two cell layers can be 
identified. In a portion of the leaf which has 50 percent of its 
eventual chlorophyll, the plastids in the two cell layers are 
different, but it is interesting that the bundle sheath cell 
chloroplasts do have grana at this stage. They are not, however, 
as well developed as the mesophyll cell chloroplasts. The grana 
in the bundle sheath cell chloroplasts in the mature cell are a 
result of reduction and are not plastids with an independent 
origin. This specialization of chloroplasts has probably followed 
cell differentiation. What we are really describing is the evo- 
lution of an amyloplast or starch-forming plastid. 

The etioplasts in dark-grown sugar cane leaves are also 
different in the two cell layers. When the dark-grown leaves are 
exposed to light, the prolamellar bodies of the respective 
etioplasts respond differently, and the bundle sheath cell plastids 
do not go through an obvious period of grana formation. Short 
exposures of light induce the etioplasts to form protrusions from 
their peripheral regions where there is an accumulation of vesicles 
and tubules. These protrusions have profiles which are very simi- 
lar to those of mitochondria. 

The C^-dicarboxylic acid pathway of photosynthetic 
fixation found in the tropical grasses has recently been demon- 
strated in members of the Amaranthaceae and Chenopodiaceae. The 
leaf anatomies of members of the genera Amaranthus and Atr iplex 
are very similar to each other and to the tropical grasses. 
Prominent bundle sheaths are surrounded by a layer of palisade 
cells. The bundle sheath cell chloroplasts are larger than the 
palisade cell chloroplasts and they store more starch, but they 
do have grana. The bundle sheath cells of both species contain 
mitochondria which are much larger than those in the palisade 
cells. The chloroplasts of these dicots possess a peripheral 
reticulum similar to that of the tropical grasses and they 
exhibit protrusions from these areas which are similar to mito- 
chondria. These features have been discussed in detail (6). 

The photosynthetic apparatus of these monocots and dicots 
have much in common and may well represent both morphological 
and biochemical convergence. There seems to be a trend towards 
the evolution of plastids in two cell layers which are special- 
ized with respect to the functions of the light and dark reaction 
of photosynthesis. It might be that there is a greater amount 
of primary fixation in the mesophyll cell chloroplasts while 
most of the starch synthesis goes on in the bundle sheath cell 
chloroplasts. This specialization of labor would result in a 
more efficient and rapid carbon fixation. There is evidence 



- 29 - 



that the plants in question evolved in tropical regions where 
aridity was a problem at some time during the year. An efficient 
system for carbon fixation would be a definite advantage in regions 
where water stress would frequently limit photosynthesis. It can 
be seen how this efficient photosynthetic system has been used by 
agriculture to great advantage in the htmiid tropics. More infor- 
mation about the adaptive significance of the behavior of the 
photosynthetic systems of tropical grasses would provide 
information which would be useful in future breeding programs. 

Literature Cited 

1. T. Bisalputra and A. Bisalputra. 1967. Occurrence of DNA 
fibrils in chloroplasts of Laurencia spectabilis . 

J. Ultrastruct. Res. 17.: 14-22. 

2. G. 0. Burr, C. E. Hartt, H. W. Brodie, T. Tanimoto, H. P. 
Kortschak, D. Takahashi, F. M. Ashton, and R. E. Coleman. 
1957, The sugar cane plant. Ann. Rev. Plant Physiol. 8^: 
275-308. 

3. M. El-Sharkawy and J. Hesketh. 1965. Photosynthesis among 
species in relation to characteristics of leaf anatomy and CO^ 
diffusion resistances. Crop Sci. 5^: 517-521, 

4. , R. S. Loomis, and W. A. Williams. 1967. 

Apparent reassimilation of respiratory carbon dioxide by 
different plant species. Physiol. Plantariom _20: 171-186. 

5. M. D. Hatch and C. R. Slack. 1966. Photosynthesis by sugar 
cane leaves. A new carboxylation reaction and the pathway of 
sugar formation. Biochem. J. 101: 103-111. 

6. W. M. Laetsch. 1968. Chloroplast specialization in dicoty- 
ledons possessing the C^-dicarboxylic acid pathway of 
photosynthetic CO2 fixation. Am. J. Botany. In press. 

7. and D. A. Stetler. 1965. Chloroplast structure 

and function in cultured tobacco tissue. Am. J. Botany 52 ; 
798-804. 

8. D. N. Moss. 1962. The limiting carbon dioxide concentration 
for photosynthesis. Nature 193 ; 587. 

9. K. Muhlethaler and A. Frey-Wyssling. 1959. Entwicklung und 
Strucktur der Plastiden. J. Biophys. Biochem. Cytol. _6: 
507-512. 



- 30 - 



10. D. von Wettstein. 1959. Developmental changes in chloro- 
plasts and their genetic control. In Developmental Biology 
(ed. , D. Rudnick) , 16th Symp. Soc . Study Develop, and Growth. 
Ronald Press, New York. 

11. Biochemistry of Chloroplasts . 1966. T. W. Goodwin (ed.), 
2 vol.. Academic Press, New York. 

12. J. T. 0. Kirk and R. A. E. Tilney-Bassett . 1967. 

The Plastids . W. H. Freeman & Company, San Francisco. 



SOME ASPECTS OF THE MOLECULAR BIOLOGY OF CHLOROPLASTS 

S. G. Wildman 

Department of Botanical Sciences and Molecular Biology Institute 
University of California, Los Angeles 

Studies of a great variety of photosynthetic organisms and 
higher plants have shown chloroplasts to contain a unique DNA which 
is different from nuclear and mitochondrial DNA (1). In tobacco, 
for example, chloroplast DNA constitutes about 10 percent of the 
total DNA in leaves, amounting to about 5 x 10""^^ g. of DNA per 
chloroplast, has a density of 1.703 vs. 1.698 for nuclear DNA, a 
minimum mol. wt . of about 4 x 10^ daltons, and in contrast to 
nuclear DNA, contains no 5-methyl cytosine, and renatures 
completely after melting (2). 

The question of what role chloroplast DNA performs in the 
development and inheritance of chloroplasts is being investigated 
by several laboratories throughout the world. In the case of 
chloroplasts from higher plants, knowledge of the nature of the 
early stages of chloroplast development is extremely scanty 
beyond the knowledge that the organelles are not present in seeds 
and therefore are not transmitted from generation to generation 
as independent, chlorophyll-containing bodies. However, chloro- 
plast DNA has been identified in seeds (3). Consequently, it is 
suspected that chloroplast DNA may be the agent which is 
transmitted and that it contains the molecular information 
necessary for the synthesis of each new generation of chloroplasts. 
Support for this view is derived from genetical evidence which has 
shown that some aspects of chloroplast development are controlled 
by extranuclear determinants (4). 



- 31 - 



Chloroplasts contained in the leaves of plants such as 
tobacco and spinach are complex organelles. When observed in 
living plant cells, they are seen to be composed of two phases: 
a stationary component containing a system of lamellae or thylakoids 
where chlorophyll is located, and a mobile phase (5). The mobile 
phase is in constant motion and exhibits curious interactions with 
the mitochondria and cytoplasmic network. Techniques have been 
developed for isolating chloroplasts with intact mobile phase (6) 
and for selectively removing the mobile phase from the lamellar 
system (7). While leaves contain both SOS and 70S ribosomes (8), 
analysis of the macromolecular composition of the mobile phase 
of chloroplasts shows it to contain the 70S ribosomes together 
with 18S Fraction I protein (carboxydismutase) and a mixture of 
4-6S proteins. The chloroplast ribosomes have physico-chemical 
and morphological properties which are closely similar to 
ribosomes obtained from microorganisms such as E. coli and blue- 
green algae (8) . 

Isolated chloroplasts are capable of incorporating amino 
acids into protein, utilizing polysomes composed of 70S ribosomes 
(9) . About one-half of the total ribosomes in chloroplasts are 
readily released with the mobile phase, whereas the remainder are 
tightly bound to the lamellar system so that the lamellae must be 
broken down by detergents before the ribosomes can be released. 
Chloroplast DNA is also tightly bound to the lamellar system. In 
addition to DNA, the lamellar system contains a DNA polymerase 
which causes Incorporation of deoxynucleoside triphosphates into 
a product identical to chloroplast DNA (10) , and an RNA polymerase 
which utilizes chloroplast DNA as a template for pol3mier ization 
of nucleoside triphosphates into RNA (11). Histones are not 
associated with the DNA and thus both the ribosomes and DNA of 
chloroplasts resemble those in bacteria and serve as an indication 
that chloroplasts are of ancient origin in the evolutionary scheme 
of things. 

Since chloroplasts appear to have all of the necessary 
enzymatic machinery which would be required for translating the 
information in chloroplast DNA into the formation of proteins, 
the question of how closely these systems are integrated with each 
other is being intensively investigated. Evidence is being sought 
as to whether chloroplasts contain specific transfer RNA's differ- 
ent from those in the cytoplasm, whether chloroplast ribosomes can 
synthesize peptides related to Fraction I protein or other enzymes 
of the photosynthetic carbon cycle, and whether chloroplast DNA 
has nucleotide sequences which could code for transfer and ribosomal 
RNA from chloroplasts. In the case of ribosomal RNA, chloroplast 
DNA hybridizes with ribosomal RNA from 70S chloroplast ribosomes 
(0.5 percent) (12,13), but not with RNA from BOS cytoplasmic 
ribosomes, the latter hybridizing with nuclear DNA (0.3 percent). 



- 32 - 



Surprisingly, chloroplast ribosomal RNA also hybridizes with 
nuclear DNA (0.1 percent). In fact, the informational content 
of nuclear DNA related to chloroplast ribosomal RNA is about 
three times greater than that contained in chloroplast DNA since 
the latter constitutes only 10 percent of the total cellular DNA. 
On a per nucleus per chloroplast basis, the information in the 
nucleus capable of coding for chloroplast RNA is about 1000 times 
greater than that contained in the DNA of a chloroplast. There 
appear to be about 800 cistrons in nuclear DNA complementary to 
chloroplast ribosomal RNA, compared to only about 8 cistrons in 
chloroplast DNA. Apparently, only about 10 percent of the chloro- 
plast DNA contains nucleotide sequences complementary to 
chloroplast ribosomal RNA suggesting that much of the potential 
information in chloroplast DNA could code for other purposes. It 
has been estimated (1) that the DNA in a single chloroplast would 
be sufficient to code for about 1600 different proteins, each 
containing 200 amino acids in the primary structure of their sub- 
units . 



Is the information in nuclei for chloroplast ribosomal RNA 
utilized during the development of chloroplasts or is it a non- 
functional relic of past evolution? There is evidence that nuclear 
and chloroplast DNA's share common nucleotide sequences as detected 
by hybridization (10,14). Perhaps the information required for a 
photosynthetic apparatus was contained in a primeval nuclear DNA 
which became separated as a distinct chloroplast DNA at the time in 
evolution when the photosynthetic apparatus became enclosed within 
an organelle distinct from the nucleus. On this basis, it would 
be no surprise to still find redundancy in the form of nuclear DNA 
sharing some base sequences in common with chloroplast DNA. How- 
ever, it seems certain that nuclear DNA does have a role in the 
development of chloroplasts as attested by the many nuclear mutants 
which affect chlorophyll biosynthesis and other aspects of 
chloroplasts (4). 



Some insight into the problem of the role of nuclear DNA in 
chloroplast development may come from investigation of a mutant 
which results in a random variegation of tobacco leaves. Cells in 
the variegated areas contain mixed populations of normal and 
defective chloroplasts, the latter being devoid of chlorophyll. 
The defective chloroplasts appear as if they were composed only 
of mobile phase with little or no lamellar systems. The defective 
chloroplasts contain a normal amount of chloroplast DNA as well as 
an active RNA polymerase, together with a normal amount of 70S 
chloroplast ribosomes. As a working hypothesis, it might be sur- 
mised that the information necessary for the macromolecular 
constituents of the mobile phase is provided by nuclear DNA. In 
this view, chloroplast DNA may contain the information necessary 
for the formation of the lamellar system, and a defect in the 



- 33 - 



chloroplast DNA may prevent synthesis and programming of a special 
class of chloroplast ribosomes required for synthesis of the lipo- 
proteins of the lamellar system. 



Literature Cited 

1. See reviews by A. Gibor and S. Granick. Science 145 : 890 
(1964); J. T. 0. Kirk. In Biochemistry of Chloroplasts 

(ed. T. W. Goodwin). Academic Press, London. Vol. 1, p. 319 
(1966). 

2. K. K. Tewari and S. G. Wildman. Science 153_: 1269 (1966). 



3. B. R. Green and M. P. Gordon. Biochlm. Biophys. Acta 145 : 378 
(1967). 

4. J. T. 0. Kirk and R. A. E. T ilney-Bassett . The Plastids . 
W. H. Freeman & Co. Ltd. (1967). 

5. S. G. Wildman. In Biochemistry of Chloroplasts (ed. T. W. 
Goodwin). Academic Press, London. Vol. 2, p. 295 (1967). 

6. S. I. Honda, T. Hongladarom, and G. G. Laties. J. Exptl. 
Botany 460 (1966) . 

7. R. 1. B. Francki, N. K. Boardman, and S. G. Wildman. 
Biochemistry _4: 865 (1965). 

8. J. W. Lyttleton. Exptl. Cell Res. 26: 312 (1962); N. K. 
Boardman, R. I. B. Francki, and S. G. Wildman. J. Mol. Biol. 
17_: 470 (1966). 

9. D. Spencer and S. G. Wildman. Biochemistry 3_= 954 (1964); 
J. L. Chen and S. G. Wildman. Science _155: 1271 (1967). 



10. K. K. Tewari and S. G. Wildman. PNAS 58_: 689 (1967); 

D. Spencer and P. R. Whitfield. B.B.R.C. 28(4): 538 (1967). 

11. J. T. 0. Kirk. B.B.R.C. 14^: 393 (1964); K. K. Tewari and 
S. G. Wildman. Fed. Proc. 26: 869 (1967); D. Spencer and 
P. R. Whitfield. Arch. Biochem. Biophys. 121: 336 (1967). 

12. N. S. Scott and R. Smillie. B.B.R.C. 28: 598 (1967). 

13. K. K. Tewari and S. G. Wildman. PNAS. In press, 1968. 

14. 0. C. Richards. PNAS 57_: 156 (1967). 



- 34 - 



BACTERIAL SPORULATION: A MODEL SYSTEM FOR THE MOLECULAR 
APPROACH TO MORPHOGENESIS 



Roy H. Doi 

Department of Biochemistry and Biophysics 
University of California, Davis 



Bacterial sporulation is an example of unicellular morpho- 
genesis in which a metabolically active, rod-shaped vegetative cell 
is converted to a dormant, ellipsoidal spore. The two forms differ 
not only in morphology and metabolic activity but in several other 
properties, including water and metal content, cell-wall structure 
and proteins, heat and chemical resistance, presence of dipicolinic 
acid, and protein and nucleic acid content. In fact, bacterial 
spores represent an interesting case of morphogenesis and an extreme 
case of biological dormancy, since they may remain dormant for at 
least 250 years. 

Sporulation is a favorable system for analyzing morphogenesis, 
because it can be approached by biochemica], cytological, and genetic 
techniques. Furthermore the different morphological stages are very 
distinct. Studies by many investigators have shown that a sequence 
of biochemical and physiological events occurs during spore forma- 
tion (1-3). Mutants have been isolated which develop to particular 
points in this sequence (4). Since RNA fractions play a key part 
in translating the genetic message during protein synthesis and may 
be involved in regulating the expression of specific genes, our 
approach 'has been to characterize the RNA fractions from vegetative 
cells, sporulating cells, and dormant spores of Bacillus subtilis . 
The goal has been to identify changes in the RNA patterns and to 
correlate them with specific functions during morphogenesis. 

Studies on bulk RNA . Sporulation occurs after active growth 
has terminated. During sporulation the net synthesis of nucleic 
acid ceases; however, an active turnover of all RNA species occurs 
(5). There is a preferential breakdown of ribosomes (6), resulting 
in an increasing ratio of transfer RNA (tRNA) to ribosomal RNA 
(rRNA) during spore formation (7). The tRNA-to-rRNA ratio reaches 
its maximum in the spore. This relationship between the tRNA and 
rRNA is related to the metabolic activity of the cell, since there 
is an inverse relationship between the tRNA/rRNA ratio and growth 
rate of B^. subtilis . The RNA content of spores is usually one-half 
that of vegetative cells. 

Studies on messenger RNA (mRNA) . If the occurrence of new 
proteins during sporulation is dependent on transcriptional control, 
mRNA synthesis should be affected. Sporulating cells contain a 
unique mRNA fraction in addition to the mRNA found in actively 



- 35 - 



growing cells. This has been shown by DNA-RNA hybrid competition 
studies (8,9). These results suggest that a general repression 
of genes active in vegetative cells does not occur; however 
specific genes are derepressed for sporulation functions. The 
fact that mRNA of germinating spores is similar to that of active 
vegetative cells indicates that genes active during sporulation 
are repressed once again during gemination. No evidence has 
been found for the presence of mRNA in dormant spores of B. 
sub til is . 

Studies on ribosomes and ribosomal RNA . Dormancy could be 
a function of some defective component in the protein-s3mthesizing 
machinery. A comparison of the ribosome fractions of spores and 
vegetative cells has revealed no significant differences in their 
physicochemical properties (10). The RNA and protein contents and 
the sedimentation properties of the ribosomes are identical. Spore 
ribosomes are able to bind synthetic polynucleotides and aminoacyl- 
tRNA to almost the same extent as vegetative cell ribosomes. The 
base composition and sedimentation coefficients of rRNA are very 
similar. DNA-rKNA hybrid competition studies have shown that rRNAs 
from spores and vegetative cells have identical or nearly identi- 
cal base sequences. These results suggest that the ribosomal 
components of the sporulating cell are enclosed intact into the 
developing spore. 

Studies on transfer RNA . Recent investigations have 
suggested that tRNAs may play a role in regulating the translation 
process. Therefore the elution profile of tRNA from vegetative 
cells and sporulating cells were compared by co-chromatography 
through a methylated albumin kieselguhr column to determine 
whether significant changes occurred in their patterns. Most of 
the aminoacyl-tRNA patterns remained constant in these analyses. 
However, a significant change in the valine tRNA pattern was 
observed in early sporulation cells (11). The alteration in the 
pattern consisted of an increase of one of the two valine tRNA 
species. A more extensive study has revealed that a similar 
alteration occurs during step-down and step-up growth transitions 
and during the stationary phase of an asporogenous mutant. We 
are investigating this change in the ratio of two valine tRNA 
species to determine whether it is due to differential synthesis 
or to some type of activation. Also it is of interest to deter- 
mine whether a direct relationship exists between tRNA pattern 
changes and sporulation or particular growth conditions. 

The tRNAs of spores are capable of accepting amino acids 
(12,13). Aninoacyl-tRNA synthetase activity has also been 
demonstrated in spore extracts. This part of the translation 
mechanism appears to be intact in dormant spores. 



- 36 - 



Summary . The sporulating bacterium Bacillus subtllis is a 
highly suitable system for analyzing RNA changes and functions dur- 
ing morphogenesis. An active turnover of KNA is noted during spore 
formation. Qualitative differences in the messenger RNA 
population are observed, indicating the expression of genes 
specific for sporulation. Changes in the pattern of valine trans- 
fer RNA during sporulation are being analyzed to determine whether 
they are due to differential synthesis or modification and whether 
they have a direct relationship to regulation of gene expression. 
Although degradation of ribosomes occurs during spore formation, 
the spore ribosomes appear to be normal. In fact all components 
of the protein synthesizing machinery of spores analyzed to date 
appear to be functional, suggesting that dormancy is not caused 
by a defect in the translation mechanism. 

Literature Cited 

1. Spores III . Eds. L. L. Campbell and H. 0. Halvorson. Am. Soc. 
Microbiol, Ann Arbor, Michigan, 1965. 

2. I. E. Young and P. C. Fitz-James. 1959. Chemical and 
morphological studies of bacterial spore formation. I. The 
formation of spores in Bacillus cereus ♦ J. Biophys. Biochem. 
Cytol. _6: 467. 

3. H. 0. Halvorson, J. C. Vary, and W. Steinberg. 1966. 
Developmental changes during the formation and breaking of the 
dormant stage in bacteria. Ann. Rev. Microbiol. 20_: 169. 

4. J. Spizizen. 1965. Analysis of asporogenic mutants in Bacillus 
subtilis by genetic transformation. In Spores III . Ed. 

L. L. Campbell and H. 0. Halvorson. Am. Soc. Microbiol., 
Ann Arbor, Mich. pp. 125-137. 

5. G. Balassa. 1966. Renouvellement des ARN et des proteines au 
cours sporulation de Bacillus subtilis . Ann. Inst. Pasteur 
110 : 316. 

6. P. 0. Fitz-James. 1963. Spore formation in wild and mutant 
strains of Bacillus cereus and some effects of inhibitors. In 
Mecanismes de Regulation des Activities Cellulaires chez les 
Microorganismes . Colloq. Interna. Centre Natl. Rech. Sci. 
(Paris), No. 124, pp. 529-544. 

7. R. H. Doi and R. T. Igarashi. 1964. Relation of ribonucleic 
acid composition to growth rate and dormancy in Bacillus 
subtillis. Nature 203: 1092. 



- 37 - 



8. R. H. Doi and R. T. Igarashi. 1964. Genetic transcription 
during morphogenesis. Proc. Natl. Acad. Sci. U.S. 52: 755. 

9. A. I. Aronson. 1965. Characterization of messenger RNA in 
sporulating Bacillus cereus . J. Molecular Biol. 11: 576. 

10. H. L. Bishop and R. H. Doi. 1966. Isolation and character- 
ization of ribosomes from Bacillus subtilis spores. 

J. Bacteriology 695. 

11. R. H. Doi, I. Kaneko, and R. T. Igarashi. 1968. Pattern of 
valine transfer ribonucleic acid of Bacillus subtilis at 
different growth conditions. J. Biol. Chem. 243 ; 947. 

12. B. Goehler and R. H. Doi. 1968. The presence and function 
of sulfur-containing transfer ribonucleic acid of Bacillus 
subtilis . J. Bacteriology 95^. In press. 

13. R. A. Lazzarini. 1966. Differences in lysine-sRNA from 
spores and vegetative cells of Bacillus subtilis . Proc. Natl. 
Acad. Sci. U.S. 56: 185. 



NUCLEIC ACIDS IN EVOLUTION, TAXONOMY AND DEVELOPMENT 

B. J. McCarthy 
Department of Microbiology, School of Medicine 
University of Washington, Seattle 

Reactions involving the formation of duplexes, either DNA/ 
DNA of DNA/RNA, are in current use in many areas of biological 
investigation. By virtue of their great specificity these reac- 
tions provide a means for comparisons of nucleotide sequences in 
nucleic acids of related organisms (1). They provide a rapid and 
convenient means for making such comparisons without the actual 
determination of base sequence. The cross-reaction of two DNA 
single strands from different organisms is a measure of the amount 
of divergence in base sequence which has taken place in the evo- 
lution of these organisms (2). This has proved useful for tracing 
the evolution of various groups of organisms from viruses to 
primates at the molecular level as well as a quantitative taxo- 
nomic method. 



- 38 - 



Similar methods are useful for the analysis of gene trans- 
cription through the formation of DNA/RNA hybrids (3). This 
methodology offers an approach to the analysis of differential 
patterns of gene expression in development (4). 



Literature Cited 

1. B. J. McCarthy. The evolution of base sequences in poly- 
nucleotides. 1965. Progress in Nucleic Acid Res. A_: 129. 

2. B. J. McCarthy. The arrangement of base sequences in 
deoxyribonucleic acid. 1967. Bacteriol. Rev. 31^: 215. 

3. D. Gillespie and S. Spiegelman. A method for the assay of 
DNA/RNA hybrids. 1965. J. Molecular Biol. 12: 829. 

4. R. B. Church and B. J. McCarthy. RNA synthesis in regener- 
ating and embryonic liver. 1967. J. Molecular Biol. 23^: 459. 



LEAF ABSCISSION: THE CHRONOLOGY AND CONTROL OF A 
TERMINAL DEVELOPMENTAL SEQUENCE 

D. James Morre 
Department of Botany and Plant Pathology 
Purdue University, Lafayette, Indiana 



Leaf abscission can be initiated by senescence, injury, 
disease, environmental changes or chemical defoliants. The develop- 
mental sequence for bean (Phaseolus vulgaris L.) leaf abscission, a 
widely used experimental system, is visualized as including: (a) 
Appearance of chemical messengers that result in induction of 
synthesis of specific informational RNA through regulation of the 
template activity in specific regions of the DNA; (b) Synthesis of 
abscission-specific enzymes, including cell-wall-dissolving 
enzjmies, under direction of the newly formed informational RNA; 
(c) Secretion of wall-dissolving enzymes into cell walls adjacent 
to the separation layer; (d) Dissolution of cell walls within the 
separation layer, which continues until some minimum-break 
strength is achieved; and (e) Separation aided by internal shear 
forces generated by differential growth and/or hydrostatic pressure. 



- 39 - 



For in vitro studies, explants (12), 1.0 to 1.5 cm. long, 
are cut from unifoliate (primary) leaves of ca. 18-day-old bean 
plants and placed upright (petiole or pulvinar end down) in 
1 percent agar in closed petri dishes. These explants contain 
the abscission zone at the leaf blade (pulvinus)-petiole juncture. 
Separation (abscission) of 50 percent of the explants occurs in 
about 100 hours. In comparison, natural abscission of unifoliate 
leaves occurs when plants are 30 to 40 days old. In vitro 
abscission experiments with plants other than bean are conducted 
in a similar manner. 

Morphological studies emphasize that cell-wall breakdown 
is an important aspect of cell separation (4,12). Bean leaf 
abscission occurs primarily by dissolution of cell contacts 
between adjacent tissue regions. 

Prior to separation layer formation, the abscission zone 
is not a point of structural weakness (9). As determined with an 
Instron linear stress-strain analyzer, formation of the separation 
layer is indicated by a marked decline in break strength across 
the abscission zone. With explants, the decline is linear for 
the first 48 to 72 hours, from a maximum of jca. 500 g at t=0 to 
a minimum of 50 to 100 g when the separation layer is complete. 

Formation of this separation layer is retarded by inhibi- 
tors of RNA (actinomycin D, 5-f luoroduracil) and protein 
(cycloheximide , puromycin) synthesis (2,3,6) and accelerated by 
ethylene and compounds that promote ethylene production (1) . As 
development proceeds, the abscission processes become less 
sensitive to inhibitors and more responsive to ethylene. 

When abscission is accelerated by ethylene, there is an 
accompanying increased rate of RNA synthesis (P-^^ incorporation) 
followed by an increased rate of protein synthesis (leucine 
incorporation) (2,3,6). Incorporation of P into all classes of 
RNA is accelerated, but the greatest increases in specific activ- 
ity are associated with the messenger fractions (6) . These changes 
in RNA and protein synthesis are restricted to the abscission zone 
and adjacent petiole-derived cell layers as observed through use 
of histochemistry and autoradiography (Barbara Webster and A. C. 
Leopold, private communication). During the 24-hour period 
following deblading and excission, patterns of H-^-leucine and 
-uridine incorporation as well as size and staining of nucleoli 
are correlated with separation layer formation and accelerated by 
application of ethylene. Affinity of nuclei for fast green (5), 
1 percent pH 8.0 to 8.1 (for basic proteins such as histones) 
increases during the first 24 hours in the pulvinus cells of bean 
explants and then remains constant (Wederitsch and Morre, 
unpublished). These observations are consistent with a "step up" 



- 40 - 



transition in the abscission zone and adjacent petiolar cells 
prior to separation layer formation and a "step down" transition 
in pulvinar cells preceding their senescence and death. 

Except for rupture of the vascular strands, wall changes 
leading to separation layer formation are complete in 72 hours. 
Chemical analyses of abscission zones reveal small quantitative 
changes (5 to 20 percent) in arabinose, galactose, and galactu- 
ronic acid of the classical pectin fraction and an increase in 
the hot- and cold-water-extractable pectin fractions at the 
expense of the pectin fraction extractable with dilute mineral 
acid (9). These results are consistent with wall dissolution 
through hydrolysis of glycosidic linkages catalyzed by the 
action of endopolygalacturonases . 



HOUR AFTER DEBLADING 24 48 72 96 120 



INDUCERS 

PULVINAR HISTONES 




RNA SYNTHESIS 1"^— ~1 



PROTEIN SYNTHESIS 
PECTIN ESTERASE 
PECTINASE 
PECTIN DISSOLUTION 
BREAK STRENGTH 
CELLULASE 
CELLULOSE DISSOL. 
DIFFERENTIAL EXP. 
RUPTURE OF STELE 
ABSCISSION 




Figure 1. Probable chronology of events during abscission of 
explants prepared from the distal abscission zone of 
unifoliate bean ( Phaseolus vulgaris ) leaves. 

The appearances of endopolygalacturonases coincides with 
wall dissolution and precedes cell separation. As determined 
by a sensitive and specific bioassay (H. W. Mussell and D. J. 
Morre, in preparation), polygalacturonase activity increases 
from no detectable enzyme at t=0 to a maximum of ca_. 0.01 yg. 
per abscission zone at 72 hours (compared with ca_. 10 yg. 
polyanhydrogalacturonic acid per abscission zone) . Results 
with neutral red staining and concomitant plasmolytic studies 

- 41 - 



suggest that the protoplasts remain intact during cell separa- 
tion and that the enzymes responsible for wall dissolution are 
secreted. Changes in pectinesterase (10,11), cellulases (7), 
and divalent ions (11) are associated with abscission in beans, 
all of which might contribute to cell-wall softening. However, 
an area of structural weakness at the abscission zone is obtained 
in vitro by treatment of half-explants with purified endopoly- 
galacturonases , and secretion of these enzymes appears both 
necessary and sufficient for the separation layer to form in 
beans. A projected chronology of abscission events in bean 
explants is summarized in figure 1. 

What molecules initiate the rise in pectinase and other 
wall dissolving enzymes? In fungi (8), pectin, galacturonic acid 
and galactose act as endopolygalacturonase inducers. When applied 
to bean explants, galactose and galacturonic acid markedly accel- 
erate abscission. The next step is to relate these effects to 
levels of enz5mies in bean cell walls. 

Literature Cited 

1. F. B. Abeles. 1967. Mechanism of action of abscission 
accelerators. Physiol. Plant. 2^: 442-454. 

2. and R. E. Holm. 1966. Enhancement of RNA syn- 
thesis, protein synthesis and abscission by ethylene. Plant 
Physiol. 41: 1337-1342. 

3. and . 1967. Abscission: Role of 

protein synthesis. Ann. N. Y. Acad. Sci. 144 : 367-373. 

4. F. T. Addicott. 1965. Physiology of abscission. Handb. 
Pf lanzenphysiol. 15: 1094-1126. 

5. M. Alfert and I. I. Geschwind. 1953. A selective staining 
method for the basic proteins of cell nuclei. Proc. Natl. 
Acad. Sci. 39: 991-999. 

6. R. E. Holm and F. B. Abeles. 1967. Abscission: The role of 
RNA synthesis. Plant Physiol. 42: 1094-1102. 

7. R. F. Horton and Daphne J. Osborne. 1967. Senescence, 
abscission and cellulase activity in Phaseolus vulgaris . 
Nature 214: 1086-1088. 

8. N. T. Keen and J. C. Horton. 1966. Induction and repression 
of endopolygalacturonase synthesis by Pyrenochaeta terrestris ♦ 
Can. J. Microbiol. 12: 443-453. 



- 42 - 



9. D. J. Morre. 1968. Cell wall dissolution and enz3mie secre- 
tion during bean leaf abscission. Plant Physiol, (in press). 

10. Daphne J. Osborne. 1958. Changes in the distribution of 
pectin methylesterase across leaf abscission zones of 
Phaseolus vulgaris . J. Exptl. Botany 9^: 446-457. 

11. H. P. Rasmussen. 1965. Chemical and physiological changes 
associated with abscission layer formation in the bean 
( Phaseolus vulgaris L. Cv. Contender). Doctoral Dissertation, 
Michigan State University. 

12. B. Rubenstein and A. C. Leopold. 1963. The nature of leaf 
abscission. Quart. Rev. Biol. _39: 356-372. 



METABOLIC AND PHYSIOLOGICAL DEVELOPMENT IN PLANT TISSUES 

George G. Laties 
Department of Botanical Sciences 
University of California, Los Angeles 

Thin slices of fleshy storage organs serve as an effective 
prototype system for biochemical ontogeny and differentiation in 
plant tissue. Immediately upon cutting, a train of biochemical 
events is evoked which results in the development of a respiratory 
increment which is qualitatively distinct from the initial, or 
basal, respiration, and which is associated with a sharp increase 
in phosphorylative activity, and a spate of physiological activity 
which depends on oxidative phosphorylation. Insofar as the trans- 
formation at issue is confined to a few layers of surface cells, 
it has been proposed that one parameter which controls the meta- 
bolic condition of a cell is its spatial relationship to its 
neighbors and to the environment. Thus, the metabolism of cells 
or tissues behind the growing point of root or shoot may change 
with the distance of the cell or tissue from the surface — and 
such appears to be the case. 

The developed respiratory rise in slices, as well as the 
associated physiological changes, are prevented by actinomycin 
and by puromycin. Whereas the respiration rise develops through 
24 hours or longer, actinomycin and puromycin inhibit the rise 



- 43 - 



only if presented during the first 10 hours. While actinomycin 
interferes with uracil incorporation into RNA, and puromycin 
inhibits leucine incorporation into protein, throughout the full 
24-hour period, what has been termed reciprocal inhibition — i.e., 
the inhibition or uracil incorporation by puromycin and leucine 
incorporation by actinomycin — is evidenced only in the first 10 
hours. The coincident time-course and extent of reciprocal inhi- 
bition evoked by puromycin and actinomycin respectively have 
suggested ribosome synthesis as the primary event leading to the 
multitude of the observable changes. Experiments wherein uracil- 

was presented in successive 4-hour intervals, and its 
incorporation into ribosomal RNA determined, have indicated that 
ribosome synthesis is marked immediately after slicing, and drops 
to a low value in 12 hours. Puromycin-sensitive RNA synthesis is 
entirely accounted for by ribosomal RNA synthesis. Thus newly 
synthesized ribosomes, and not new messenger RNA alone, underlie 
the metabolic changes which follow slicing. 

The metabolic block in fresh slices appears to center on 
isocitric dehydrogenase. However, while fresh slices show no 
overall tricarboxylic acid cycle activity, mitochondria from fresh 
tissue as normally isolated implement each step of the cycle. 
Methods have been developed to prepare mitochondria morphologi- 
cally akin to mitochondria in vivo, with the end in view of 
ascertaining whether mitochondria from fresh tissue, which have 
not been altered morphologically during isolation, display a 
relatively inactive isocitric dehydrogenase. Filiform mitochon- 
dria have been prepared by the addition of any of a number of 
large-molecular-weight substances to the preparative medium, and 
such filiform mitochondria are reversibly transformable to 
spherical forms, without swelling. An examination of the isotherm 
for isocitrate oxidation by both filiform and spherical mitochon- 
dria, from fresh and aged tissue, points to the likelihood that 
mitochondrial isocitric dehydrogenase is under allosteric control 
in potato. It remains to establish with certainty whether the 
ultimate consequence of the complex and extensive biochemical 
events which are initiated by slicing is the unfettering of iso- 
citric dehydrogenase. 



- 44 - 



REFERENCES 



G. G. Laties. 1963. Control of respiratory quality and 

magnitude during development. In Control Mechanisms 
in Respiration and Fermentation . B. Wright, Ed., 
Ronald Press, New York, pp. 129-155. 

. 1964, The onset of tricarboxylic acid cycle 

activity with aging in potato slices. Plant Physiol. 
39: 654-663. 

. 1965. Inhibition of RNA and protein synthesis 

by chloral in potato slices. Plant Physiol. 40: 1237- 
1241. 

M. Sampson and G. G. Laties. 1968. Ribosomal RNA synthesis in 
newly sliced discs of potato tuber. Plant Physiol. 
In press. 

T. Treffry and G. G. Laties. 1968. Reversible morphological 
transformation of unswollen potato mitochondria. 
J. Cell Science. Submitted. 



ATTENDANCE 

(Asterisks indicate representatives of the 12 Western State 
Agricultural Experiment Stations who served as collaborators.) 



Douglas R. Black 
Graduate Student 
Univ. of Calif., Berkeley 

Rosalinda Boasson 

Graduate Student 

Univ. of Calif. , Berkeley 

James Bonner 

Professor of Biology 

Calif. Institute of Technology 

Pasadena 

*John A. Booth 
New Mexico State University 
Las Cruces 

R. W. Breidenbach 
Univ. of Calif. , Davis 



George Bruening 

Univ. of Calif., Davis 

David Cole 

Univ. of Calif. , Berkeley 

Barbara Cooper, Graduate Student 
Univ. of Calif. , Berkeley 

Donald A. Corlett, Jr. 
Del Monte Corporation 
San Francisco 

Mrs. Koose Daley 

Univ. of Calif. , Berkeley 

Arthur DeVries 

Univ. of Calif. , Davis 



- 45 - 



Fred J. Dill 

Graduate Student 

Univ. of Calif. , Berkeley 

Roy H. Doi 

Univ. of Calif., Davis 

Karl Drlica, Student 
Univ. of Calif. , Berkeley 

Murray E. Duysen 

North Dakota State University 

Fargo 

R. M. Endo 

Univ. of Calif. , Riverside 

*Harold J. Evans 
Oregon State Univ. , Corvallis 

Robert E. Feeney 
Univ. of Calif., Davis 

Philip Filner 
Michigan State Univ., 
East Lansing 

*Dean C. Fletcher 
Univ. of Nevada, Reno 

Earl Fronk 

Univ. of Calif., Berkeley 

Jonathan Goldthwaite 

Graduate Student 

Univ. of Calif. , Berkeley 

Shirl 0. Graham 
Washington State Univ. 
Pullman 

Reed A. Gray 

Stauffer Chemical Company 
Mt. View, Calif. 

Frank C. Greene, Chemist 
Univ. of Calif., Davis 



Lee A. Hadwiger 

Washington State University 

Pullman 

"Erhardt R. Hehn 
Montana State University 
Bozeman 

Theodore C. Hsiao 
Univ. of Calif., Davis 

R. C. Huffaker 

Univ. of Calif., Davis 

Keith A. Ito 

National Canners Association 
Berkeley, Calif. 

Barbara W. Jansen 

Univ. of Calif., Berkeley 

Jean Jansen 

Cutter Laboratories, Berkeley 

Russell L. Jones 

Univ. of Calif., Berkeley 

Tom Jukes 

Univ. of Calif. , Berkeley 

Clarence I. Kado 

Univ. of Calif., Berkeley 

Jurgen Koch 

Univ. of Calif., Berkeley 

Joseph S. Krakow 

Univ. of Calif. , Berkeley 

*N. P. Kef ford 
Univ. of Hawaii, Honolulu 

C. Arthur Knight 

Univ. of Calif. , Berkeley 

Stanley Komatsu 

Univ. of Calif., Davis 



- 46 - 



W. M. Laetsch 

Univ. of Calif. , Berkeley 

George Laties 

Univ. of Calif., Los Angeles 

Joseph Ludlow 

Univ. of Calif., Berkeley 

Harold Martinson 
Graduate Student 
Univ. of Calif. , Berkeley 

John T. Marvel 

Univ. of Arizona, Tucson 

Brian J. McCarthy 

Univ. of Washington, Seattle 

''Dr. Vern McMahon 
Univ. of Wyoming, Laramie 

D. James Morre 

Purdue Univ., Lafayette, Ind . 

Charles W. Nagel 

Washington State Univ., Pullman 

Edward A. Norberg 

Graduate Student 

Univ. of Calif. , Berkeley 

Ferenc M. Pallos 
Stauffer Chemical Co. 
Richmond, Calif. 

*J. R. Ridley 
Univ. of Idaho, Moscow 

Roger Romani 

Univ. of Calif., Davis 

*Cleon W. Ross 
Colorado State University 
Fort Collins 



W. S. Ruliffson 

Kansas State University 

Manhattan 

C. H. Ryan 

Washington State University 
Pullman 

Martin E. Schwochau 
Washington State University 
Pullman 

J. S. Semancik 

Univ. of Calif., Riverside 

R. J. Shepherd 

Univ. of Calif., Davis 

James J. Sims 

Univ. of Calif., Riverside 

'^Rex S. Spendlove 
Utah State Univ. , Logan 

*P. K. Stumpf 
Univ. of Calif. , Davis 

Jwe Sheng Tung 

Graduate Student 

Univ. of Calif., Berkeley 

Edwin G. Wallace 
Stauffer Chemical Co. 
Richmond, Calif. 

John R. Whitaker 
Univ. of Calif. , Davis 

S. G. Wildman 

Univ. of Calif. , Los Angeles 



- 47 - 



From Western Utilization Research and Development Division 
U.S. Department of Agriculture, Albany, California 



D. Althausen 
John E. Amoore 
Henry Bayne 
Dale R. Black 
Lois E. Boggs 
Kathryn Caldwell 
Michael J. Copley 
W. C. Dietrich 
Robert V. Enochian 
John Evans 
Bernard Feinberg 
Ronald Fields 
Bernard J. Finkle 
J. G. Fullington 
W. Gaffield 
John A. Garibaldi 
L. F. Ginnette 
Alan E. Goodban 
Dante G. Guadagni 
Jack Guggolz 
M. Gumbmann 
Earl Hautala 
David F. Houston 



Eugene F. Jansen 
Donald Kasarda 
Randy Knox 
Ralph Kurtzman 
Don Kuzmicky 
Laurence L. Layton 
Margaret Li 
H. Lineweaver 
A. L. Livingston 
Robert Lundin 
C. K. Lyon 
M. S. Masri 
Rachel Makower 
R. McCready 
John J. Meehan 
Emory Menefee 
H. David Michener 
J. C. Miers 
Ali Mohammad 
Albert Mossman 
Carolyn Nelson 
Harry Neumann 
Henry Ng 



C. C. Nimmo 
George K. Notter 
Marvel-Dare Nutting 
Alfred C. Olson 
Robert L. Olson 
Kenneth J. Palmer 
Rhoda Palter 

E. L. Pippen 
Allen Pittman 
Russell T. Prescott 
Clyde Rasmussen 

D. Reznick 
Sigmund Schwimmer 
William L. Stanley 
Benjamin Stark 
Dirk Stigter 

Fred Stitt 
John 0. Thomas 
Joan Wallace 
Glenn Watters 
Ed Wheeler 
Chuen-Shang Wu 
James C. Zahnley 



- 48 - 



GPO 976-670 



UNITED STATES DEPARTMENT OF AGRICULTURE 
AGRICULTURAL RESEARCH SERVICE 
Western Utilization Research a Development Division 
800 BUCHANAN STREET 
ALBANY. CALIFORNIA 947 1 O 



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